interactions between energy transducer tonb and...

234
Interactions between energy transducer TonB and ferric hydroxamate transport proteins from Escherichia coli by David M. Carter Department of Microbiology and Immunology McGill University, Montreal August 2009 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © David M. Carter, 2009

Upload: lekiet

Post on 30-Jul-2018

217 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

Interactions between energy

transducer TonB and ferric

hydroxamate transport proteins from

Escherichia coli

by

David M. Carter

Department of Microbiology and Immunology

McGill University, Montreal

August 2009

A thesis submitted to McGill University in partial fulfillment of the requirements

of the degree of Doctor of Philosophy

© David M. Carter, 2009

Page 2: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

ii

Abstract

Ph.D. David M. Carter Department of Microbiology and Immunology

The ferric hydroxamate uptake (Fhu) system of Escherichia coli transports

ferric hydroxamate-type siderophores. This system comprises outer membrane

(OM) receptor FhuA, periplasmic binding protein FhuD, and ABC permease

FhuB/C. In addition, transport through FhuA requires energy input provided by

the cytoplasmic membrane (CM)-embedded TonB/ExbB/ExbD multi-protein

complex.

This thesis focuses on identification and characterization of protein–

protein interactions that facilitate siderophore transport. Phage display

technology predicted protein–protein interactions involved in TonB-dependent

transport; peptide motifs predicted to bind TonB were identified from phage

panning experiments using purified TonB. Peptide sequences that were displayed

on TonB-interacting phage were similar to sequences of periplasm-exposed

regions on FhuA and therefore predicted FhuA regions that TonB might bind to.

Binding to these regions was confirmed by ELISA; predicted TonB-binding

sequences were fused to maltose-binding protein (MBP) and binding to TonB was

confirmed by immunoreactivity towards monoclonal antibodies directed against

MBP.

TonB was also found to bind FhuD. Peptide sequences displayed on

TonB-binding phage identified regions within FhuD to which TonB was predicted

to bind. Furthermore, phage panning experiments against purified FhuD

predicted complementary regions on TonB that FhuD was predicted to bind.

Page 3: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

iii

Binding between TonB and FhuD was confirmed in vitro. Accordingly,

biophysical methods confirmed that TonB and FhuD formed a 1:1 siderophore-

independent complex with an affinity (KD) ranging from 20-200 nM.

Further analyses demonstrated that regions proximal to TonB’s C-

terminus were essential for interaction with FhuD. Binding was characterized

between FhuD and three periplasmic TonB derivatives: a derivative possessing

residues 33–239, a derivative with a deletion of TonB’s central proline-rich

region, and a derivative possessing residues 103–239. Surface plasmon resonance

technology confirmed that all derivatives bound FhuD in concentration-dependent

manners with similar low nanomolar affinities. TonB-derived oligopeptides that

were predicted to bind FhuD were computationally docked to a FhuD crystal

structure. Docking solutions suggested that, when bound to TonB in vivo, FhuD’s

siderophore binding site would orient towards the OM where it could bind

siderophore as it emerges from FhuA’s lumen during transport. These findings

increase our knowledge of TonB-dependent transport by delineating regions of

interaction between protein partners of a siderophore transport system.

Page 4: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

iv

Résumé

Ph.D. David M. Carter Département de microbiologie et d’immunologie

Le transport des sidérophores de type hydroxamique de la bactérie

Escherichia coli est effectué par le système d’acquisition du fer hydroxamique

(Fhu) qui comprend FhuA, le récepteur de la membrane externe, FhuD, une

protéine périplasmique de liaison et FhuB/C, une perméase de type ABC. De

plus, pour effectuer le transport, FhuA requiert de l’énergie qui lui est fournie par

le complexe de protéines TonB/ExbB/ExbD situé dans la membrane

cytoplasmique.

Cette thèse se concentre sur l’identification et la caractérisation des

intéractions protéine–protéine impliquées dans le transport des sidérophores. La

technique de phage display, en déterminant des motifs peptidiques qui se lient à

la protéine TonB, a permis d’identifier des intéractions protéine–protéine

impliquées dans le transport TonB-dépendant. Les séquences peptidiques de

phage qui se sont liées à la protéine TonB correspondent à des régions de FhuA

exposées au périplasme, ce qui permet d’avancer que les deux protéines entrent en

contact en ces endroits. Pour confirmer ces résultats, les séquences peptidiques

présumées se lier à TonB ont été fusionnées à la protéine de liaison du maltose

(MBP) pour être testées contre TonB par ELISA. L’immunoréaction de ces

constructions avec des anticorps monoclonaux contre MBP ont permis de

confirmer ces intéractions.

TonB se lie aussi à FhuD. La séquence peptidique des phages qui ont liés

TonB correspond à certaines régions de la protéine FhuD. De plus, l’utilisation de

phage display contre FhuD a permis de déterminer une autre série de séquences

peptidiques qui correspondent à la protéine TonB. Ensemble, ces deux séries de

Page 5: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

v

séquences peptidiques identifient des sites d’intéractions complémentaires entre

FhuD et TonB. Des méthodes biophysiques ont permis de confirmer l’intéraction

entre TonB et FhuD in vitro. Celles-ci montrent la formation d’un complexe

ayant une stœchiométrie de liaison de 1:1 ayant une affinité (KD) se situant entre

20 et 200 mM et qui est indépendante de la présence d’un sidérophore.

Des analyses plus poussées ont permis de démontrer que la région la plus

proche de l’extrémité carboxy-terminale de TonB était essentielle pour

l’intéraction avec FhuD. Les caractéristiques de liaison entre FhuD et trois

différentes constructions de TonB ont été étudiées: une construction incluant les

acides aminés 33 à 239, une construction dénuée de la partie centrale riche en

proline et une construction comprenant les acides aminés 103 à 239. La technique

de résonance plasmonique de surface (SPR) a permis de confirmer l’intéraction

entre FhuD et les trois différentes constructions comme étant dépendante de leur

concentration et ayant un niveau d’affinité similaire d’ordre nanomolaire.

L’intéraction entre FhuD et les séquences de peptides de TonB prédites a

été étudié par simulation informatique d’ancrage avec la structure cristalline de

FhuD. Les résultats obtenus suggèrent que lorsque FhuD se lie à TonB in vivo, il

se positionne de façon à ce que le site de liaison du sidérophore s’oriente vers la

membrane externe, d’où il pourrait se lier avec le sidérophore dès que celui-ci

quitte la cavité de FhuA.

Ces résultats améliorent notre compréhension du transport TonB-dépendant en

cernant plus précisément les régions d’intéraction entre les différentes protéines

impliquées dans l’acquisition des sidérophores.

Page 6: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

vi

Acknowledgements

First, I thank my supervisor, Dr. James Coulton, for providing the

opportunity to study in a stimulating and supportive lab environment. His

encouragement as an active participant in any activity has always extended far

beyond the pursuit of science. While I can confidently say that I have learned

many techniques that will enhance my career as a scientist, I have also learned

other valuable lessons. My abilities to critically think, write and verbally

communicate have all been finely crafted during my training in the Coulton lab.

For these experiences and many more I am grateful.

I am also thankful to members of my Advisory Committee: Dr. Greg De

Crescenzo, Dr. Hervé LeMoual, and Dr. Allan Matte for their helpful advice and

critical evaluation of my academic progress.

I thank members of the Coulton lab, both past and present, who have been

constant sources of encouragement as well as sources of the often needed

diversion. Our conversations about science have always reassured my career

choice, while our social antics have made me feel all that more connected to a

supporting and caring community. To me, members of the Coulton lab have

always been more than just my associates; they are my friends for life.

My wife, Leila, has also been a constant source of support. I will always

be thankful that our shared passions of music, art and fine dining as well as our

mutual desire for adventure never grow old. I am also thankful that our shared

senses of humor are keeping us so young at heart.

I wish to thank the rest of my family, especially my parents, Orangie and

Percy, who deserve far more thanks and appreciation than there is space to write

in this thesis. With unconditional support, they have always been by my side and

have guided me through some of the most difficult decisions I’ve had to make. I

thank them for supporting all of these decisions.

Finally, I wish to thank all of my friends in Montreal, both those who live

here now and those who once did. I especially thank Jacek Stolcman, Rebecca

McTavish and Amy Gowertz for introducing me to everyone here in Montreal and

for making me realize that I have a family here in this wonderful city.

Page 7: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

vii

Contributions to original knowledge

1. Use of phage display technology to predict TonB-binding surfaces on the

periplasmic surfaces of FhuA, FepA, FecA and BtuB.

2. Demonstration that FhuA-derived, TonB-binding peptide sequences bound to

TonB in vitro. In doing so, this was the first literature report on the use of phage

display to predict bacterial protein–protein interactions.

3. Use of phage display technology to predict that TonB and FhuD would interact

and localized complementary regions of binding between these two proteins.

4. Demonstration that TonB and FhuD interact in vitro and that complementary

regions of interaction between these proteins localize to where they were

predicted.

5. Determination of the stoichiometry and affinity of TonB–FhuD interactions.

6. Demonstration that TonB, FhuA and FhuD can form a ternary complex.

7. Identification of essential regions of interaction between TonB and FhuD.

8. Prediction of the mode of binding between TonB and FhuD.

9. Generation of TonB–FhuD and TonB–FhuA–FhuD computational models.

Page 8: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

viii

Contributions of Authors

Chapter 2: Carter DM, Gagnon JN, Damlaj M, Mandava S, Makowski L,

Rodi DJ, Pawelek PD and Coulton JW. (2006) Phage display reveals multiple

contact sites between FhuA, an outer membrane receptor of Escherichia coli, and

TonB. Journal of Molecular Biology 357(1): 236-251.

David Carter: Purified TonB. Amplified TonB-affinity-selected phage.

Purified and sequenced TonB-affinity-selected phage DNA. Performed global

analyses on TonB affinity-selected peptides. Performed ELISA with purified

TonB, MBP fusion proteins and FhuA. Wrote manuscript.

Jean-Nicolas Gagnon: Panned phage libraries against TonB. Amplified TonB-

affinity selected phage. Purified and sequenced TonB-affinity-selected phage

DNA.

Moussab Damlaj: Panned phage libraries against TonB. Amplified TonB-

affinity selected phage. Purified and sequenced TonB-affinity-selected phage

DNA. Cloned and purified MBP fusions.

Suneeta Mandava, Dr. Lee Makowski and Dr. Diane Rodi: Assisted in RELIC

analyses.

Page 9: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

ix

Dr. Peter Pawelek: Performed RELIC analyses and bioinformatic analyses on

TonB-affinity-selected peptides. Performed computational docking experiments.

Wrote relevant sections of manuscript.

Chapter 3: Carter DM, Miousse IR, Gagnon JN, Martinez É, Clements A,

Lee J, Hancock MA, Gagnon H, Pawelek P and Coulton JW. (2006)

Interactions between TonB from Escherichia coli and the periplasmic protein

FhuD. Journal of Biological Chemistry 281(46):35413-35424.

David Carter: Amplified TonB-affinity-selected phage. Purified and sequenced

TonB-affinity-selected phage DNA. Performed RELIC analyses on TonB

affinity-selected peptides. Assisted with TonB and FhuD purifications.

Generated and purified FhuD T181C mutant. Fluorescently labeled FhuD.

Performed fluorescence titrations with TonB and FhuD. Generated computational

TonB–FhuA–FhuD ternary complex model. Wrote manuscript.

Isabelle Racine-Miousse: Panned phage libraries against FhuD. Amplified

FhuD-affinity selected phage. Purified and sequenced FhuD-affinity-selected

phage DNA.

Jean-Nicolas Gagnon: Panned phage libraries against TonB. Amplified TonB-

affinity selected phage. Purified and sequenced TonB-affinity-selected phage

DNA.

Page 10: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

x

Éric Martinez: Performed preliminary TonB–FhuD interaction experiments

Abigail Clements: Assisted with material preparation for TonB–FhuA–FhuD

multicomponent SPR analyses.

Ryan Jongchan Lee: Assisted with TonB and FhuD purifications.

Dr. Mark Hancock: Performed and analyzed SPR experiments. Wrote relevant

sections of manuscript.

Dr. Hubert Gagnon: Performed and analyzed DLS experiments. Wrote relevant

sections of manuscript.

Dr. Peter Pawelek: Performed RELIC analyses and bioinformatic analyses on

TonB-affinity-selected peptides. Wrote relevant sections of manuscript.

Page 11: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xi

Chapter 4: Carter DM, Deme JC, Hancock MA and Coulton JW. C-terminal

region of TonB positions periplasmic binding protein FhuD for siderophore

transport in Escherichia coli. Submitted to Protein Science.

David Carter: Generated TonB 103–239 derivative. Purified TonB 103–239

and FhuD. Analyzed AUC data. Collected and analyzed fluorescence data.

Performed computational modeling experiments. Performed bioinformatic

analyses. Wrote manuscript.

Justin Deme: Purified TonB Δ66–100. Analyzed AUC data.

Dr. Mark Hancock: Performed and analyzed SPR experiments. Wrote relevant

sections of manuscript.

Page 12: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xii

Table of contents

Abstract...................................................................................................................ii

Résumé...................................................................................................................iv

Acknowledgements...............................................................................................vi

Contributions to original knowledge..................................................................vii

Contributions of authors....................................................................................viii

List of figures........................................................................................................xx

List of tables....………………………………………………………………..xxiii

Chapter 1: Literature review and thesis objectives

1.0 Role of iron in the bacterial life cycle.............................................................2

1.0.1 Toxicity of iron................................................................................................2

1.0.2 Limitations of iron bioavailability..................................................................3

1.0.3 Bacterial iron sources.....................................................................................4

1.1 Gram-negative cell envelope...........................................................................4

1.1.1 Outer membrane.............................................................................................5

1.1.2 Lipopolysaccharide.........................................................................................5

1.1.3 Inner leaflet of outer membrane......................................................................6

1.1.4 Outer membrane proteins...............................................................................6

1.1.5 Periplasm........................................................................................................8

1.1.6 Peptidoglycan..................................................................................................8

1.1.7 Lipoproteins..................................................................................................10

1.1.8 Periplasmic proteins.....................................................................................10

Page 13: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xiii

1.1.9 Cytoplasmic membrane.................................................................................11

1.2 Nutrient transport across the cell envelope.................................................11

1.2.1 Passive transport...........................................................................................12

1.2.2 Facilitated transport.....................................................................................12

1.2.3 Active transport.............................................................................................14

1.3 General bacterial strategies for iron acquisition.........................................15

1.3.1 Ferrous iron transport..................................................................................15

1.3.2 Ferric iron transport.....................................................................................16

1.4 Siderophores...................................................................................................17

1.4.1 Siderophore biosynthesis..............................................................................20

1.4.2 Siderophore secretion...................................................................................20

1.5 TonB-dependent transporters.......................................................................21

1.5.1 Structures of TonB-dependent transporters..................................................21

1.5.2 Ton boxes......................................................................................................23

1.5.3 Transcriptional regulatory domains.............................................................24

1.5.4 Siderophore-induced conformational changes.............................................25

1.6 The TonB–ExbB–ExbD complex..................................................................29

1.6.1 TonB: Energy transducer..............................................................................30

1.6.2 TonB proline-rich region..............................................................................31

1.6.3 TonB C-terminal regions form a compact structure.....................................32

1.6.4 Oligomeric state of TonB..............................................................................32

1.7 Protein–protein interactions involving TonB..............................................34

1.7.1 TonB–ExbB–ExbD interactions....................................................................34

Page 14: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xiv

1.7.2 TonB–OM receptor interactions...................................................................36

1.7.3 TonB–cell envelope protein interactions......................................................39

1.8 Mechanisms of TonB–dependent energy transduction..............................40

1.8.1 The shuttle model..........................................................................................40

1.8.2 The rotation model........................................................................................42

1.8.3 The mechanical pulling model......................................................................44

1.8.4 Conformational rearrangement of TBDT cork domains...............................46

1.9 Periplasmic siderophore transport...............................................................47

1.9.1 Ligand-induced periplasmic binding protein conformational changes........48

1.10 Cytoplasmic membrane permeases............................................................50

1.10.1 Permease–periplasmic binding protein interactions..................................51

1.10.2 Permeases: transport mechanism...............................................................52

1.10.3 Intracellular fate of iron.............................................................................53

1.11 Introduction to techniques used in this thesis...........................................54

1.11.1 Phage display..............................................................................................54

1.11.2 Surface plasmon resonance........................................................................57

1.11.3 Dynamic light scattering.............................................................................58

1.11.4 Analytical ultracentrifugation.....................................................................59

1.12 Rationale and thesis objectives...................................................................60

Preface to chapter 2.............................................................................................62

Page 15: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xv

Chapter 2: Phage display reveals multiple contact sites

between FhuA, an outer membrane receptor of

Escherichia coli, and TonB

2.0 Summary.........................................................................................................64

2.1 Introduction....................................................................................................65

2.2 Materials and methods..................................................................................70

2.2.1 Bacterial strains and media..........................................................................70

2.2.2 Chemicals and reagents................................................................................70

2.2.3 Protein purification.......................................................................................71

2.2.4 Phage M13 titre............................................................................................71

2.2.5 Panning procedures......................................................................................72

2.2.6 Isolation of phage M13 clones, DNA isolation and sequencing...................73

2.2.7 Global analysis of affinity-selected peptides................................................73

2.2.8 Cloning of peptide-coding DNA sequences into pMal-pIII vector...............74

2.2.9 Peptide-MBP expression...............................................................................74

2.2.10 Enzyme linked immunosorbent assay (ELISA)............................................75

2.3 Results.............................................................................................................76

2.3.1 Isolation of affinity-selected peptides by phage panning..............................76

2.3.2 Global analysis of affinity-selected peptides................................................77

2.3.3 Identification of TonB-binding sites on the periplasmic surface of FhuA....80

2.3.4 Identification of potential TonB-binding sites in structurally conserved OM

receptors.................................................................................................................85

Page 16: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xvi

2.3.5 Interactions of TonB and [FhuA peptide-MBP] fusion proteins in vitro......91

2.4 Discussion........................................................................................................93

2.5 Acknowledgements.......................................................................................101

Preface to chapter 3...........................................................................................102

Chapter 3: Interactions between TonB from Escherichia

coli and the periplasmic protein FhuD

3.0 Summary.......................................................................................................104

3.1 Introduction..................................................................................................105

3.2 Materials and methods................................................................................109

3.2.1 Bacterial strains, phage libraries, and media.............................................109

3.2.2 Chemicals and reagents..............................................................................109

3.2.3 Protein purification.....................................................................................110

3.2.4 Phage display..............................................................................................111

3.2.5 Dynamic light scattering.............................................................................111

3.2.6 Fluorescence spectroscopy.........................................................................113

3.2.7 Surface plasmon resonance (SPR)..............................................................115

3.3 Results...........................................................................................................117

3.3.1 Identification of TonB-binding sites on FhuD by phage display................117

3.3.2 Identification of FhuD-binding sites on TonB by phage display................118

3.3.3 Detection of a TonB–FhuD complex by dynamic light scattering..............126

3.3.4 Detection of a TonB–FhuD complex by fluorescence spectroscopy...........127

Page 17: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xvii

3.3.5 Detection of a TonB–FhuD complex by surface plasmon resonance.........132

3.4 Discussion......................................................................................................135

3.5 Acknowledgements.......................................................................................141

Preface to chapter 4...........................................................................................143

Chapter 4: C-terminal region of TonB positions

periplasmic binding protein FhuD for siderophore

transport in Escherichia coli

4.0 Summary.......................................................................................................145

4.1 Introduction..................................................................................................146

4.2 Materials and methods................................................................................149

4.2.1 Bacterial strains and plasmids....................................................................149

4.2.2 Cloning of TonB 103–239...........................................................................149

4.2.3 Protein expression.......................................................................................149

4.2.4 Protein purifications...................................................................................150

4.2.5 Analytical ultracentrifugation.....................................................................151

4.2.6 Fluorescence spectroscopy.........................................................................151

4.2.7 Surface plasmon resonance........................................................................152

4.2.8 Computational docking...............................................................................153

4.3 Results...........................................................................................................154

4.3.1 TonB derivatives are elongated monomers with similar elements of tertiary

structure...............................................................................................................154

Page 18: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xviii

4.3.2 TonB derivatives bind FhuD with equal affinities......................................157

4.3.3 Computational models predict the orientation of FhuD when bound to

TonB.....................................................................................................................158

4.4 Discussion......................................................................................................161

4.5 Acknowledgments........................................................................................168

Preface to chapter 5...........................................................................................169

Chapter 5: Preliminary crystallization of the TonB–FhuD

complex

5.1 Introduction..................................................................................................171

5.2 Materials and methods................................................................................172

5.2.1 Bacterial strains and plasmids....................................................................172

5.2.2 Protein expression and purification............................................................172

5.2.3 Removal of FhuD His-tag...........................................................................172

5.2.4 TonB–FhuD–Fcn complex formation.........................................................173

5.2.5 TonB–FhuD–Fcn crystallization screening................................................174

5.2.6 Assessing TonB degradation.......................................................................174

5.2.7 TonB–FhuD cross-linking...........................................................................175

5.3 Results...........................................................................................................175

5.3.1 Protein preparations and processing..........................................................175

5.3.2 TonB–FhuD–Fcn complex formation.........................................................176

5.3.2 High-throughput crystallization screening.................................................176

Page 19: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xix

5.3.3 TonB exhibits time-dependent degradation................................................179

5.3.4 TonB–FhuD formaldehyde-cross-linking...................................................181

5.4 Discussion......................................................................................................182

5.5 Acknowledgements.......................................................................................184

Chapter 6: Conclusions and future work

6.0 Thesis objectives within the context of TonB-dependent transport........186

6.1 Directions for future research.....................................................................188

6.1.1 Demonstration of TonB–FhuD interactions in vivo....................................188

6.1.2 Refinement of TonB–FhuD interaction localizations..................................189

6.1.3 TonB–FhuD crystallization.........................................................................190

6.1.4 Phage display predictions of TonB-interacting proteins............................190

6.1.5 Determination of whether TonB regulates binding of siderophore to

FhuD....................................................................................................................191

6.1.6 Elucidation of siderophore binding sites by phage display........................191

References.....................................................................................................193

Page 20: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xx

List of Figures

Chapter 1

Figure 1.1. Gram-negative bacterial cell envelope.................................................7

Figure 1.2. E. coli OM nutrient transporters.........................................................13

Figure 1.3. Siderophores transported by E. coli....................................................19

Figure 1.4. Structures of TonB-dependent transporters........................................23

Figure 1.5. FhuA exhibits periplasmic conformational changes upon binding

ferrichrome.............................................................................................................26

Figure 1.6. The TonB–ExbB–ExbD complex.......................................................31

Figure 1.7. Structures of C-terminal, E. coli TonB derivatives............................33

Figure 1.8. Crystal structures of TonB bound to FhuA and BtuB........................38

Figure 1.9. Shuttle model of TonB-dependent energy transduction.....................41

Figure 1.10. Rotation model of TonB-dependent energy transduction.................43

Figure 1.11. Mechanical pulling model of TonB-dependent energy

transduction............................................................................................................45

Figure 1.12. Periplasmic binding proteins............................................................48

Figure 1.13. Phage panning...................................................................................56

Chapter 2

Figure 2.1. Alignments of affinity-selected peptides to FhuA as identified by

RELIC/MATCH.....................................................................................................83

Page 21: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xxi

Figure 2.2. RELIC/HETEROalign similarity scores mapped to the periplasm-

exposed surface of FhuA (PDB code 2FCP).........................................................86

Figure 2.3. Alignments of affinity-selected peptides to the Ton box regions and

periplasm-exposed turns of BtuB, FecA and FepA as identified by

RELIC/MATCH.....................................................................................................89

Figure 2.4. RELIC/HETEROalign similarity scores mapped to the periplasm-

exposed surfaces of BtuB (PDB code 1NQE), FecA (PDB code 1KMO), and

FepA (PDB code 1FEP).........................................................................................90

Figure 2.5. Interactions of TonB and [FhuA-MBP] fusion proteins in vitro........92

Figure 2.6. TonB-binding surfaces on the periplasmic face of FhuA...................98

Chapter 3

Figure 3.1. Alignments of TonB affinity-selected peptides to FhuD as identified

by RELIC/MATCH..............................................................................................121

Figure 3.2. TonB-binding regions identified by phage display mapped to FhuD

(PDB code 1EFD)................................................................................................122

Figure 3.3. Alignments of FhuD affinity-selected peptides to TonB..................124

Figure 3.4. FhuD-binding region identified by phage display mapped to TonB

(PDB code 1XX3)................................................................................................125

Figure 3.5. Binding of Fcn to FhuD and to FhuD T181C...................................129

Figure 3.6. Binding of TonB to AEDANS-labeled FhuD T181C and to MDCC-

labeled FhuD T181C............................................................................................131

Page 22: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xxii

Figure 3.7. Real-time kinetics of TonB–FhuD binding interaction detected by

SPR.......................................................................................................................134

Figure 3.8. Multicomponent SPR analysis to detect ternary complex formation

between FhuA–TonB–FhuD................................................................................135

Figure 3.9. Model of a FhuA-TonB-FhuD ternary complex...............................140

Chapter 4

Figure 4.1. Schematic representations of TonB derivatives from this study......155

Figure 4.2. Single cycle kinetic analysis of FhuD binding to TonB derivatives

using label-free, real-time SPR............................................................................157

Figure 4.3. TonB region II peptide docked to the surface of FhuD....................160

Figure 4.4. Sequence conservation of FhuD from various pathogenic Gram-

negative bacteria..................................................................................................166

Chapter 5

Figure 5.1. Protein purification and processing..................................................177

Figure 5.2. Complexation of TonB 103–239–FhuD–Fcn...................................177

Figure 5.3. Crystallization screen of the TonB 103–239–FhuD–Fcn

complex................................................................................................................178

Figure 5.4. Degradation of TonB 103–239.........................................................180

Figure 5.5. Formaldehyde cross-linking of TonB–FhuD complex.....................181

Page 23: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xxiii

List of Tables

Chapter 2

Table 2.1. RELIC/MATCH identification of TonB-affinity-selected Ph.D.-12

peptides corresponding to FhuA sequences...........................................................84

Table 2.2. RELIC/MATCH Identification of TonB-affinity-selected Ph.D.-C7C

peptides corresponding to FhuA sequences...........................................................84

Table 2.3. RELIC/MATCH Identification of TonB-affinity-selected Ph.D.-12

peptides corresponding to BtuB, FecA, and FepA sequences...............................87

Table 2.4. RELIC/MATCH Identification of TonB-affinity-selected Ph.D.-C7C

peptides corresponding to BtuB, FecA, and FepA sequences...............................87

Chapter 3

Table 3.1. RELIC/MATCH identification of TonB-affinity-selected Ph.D.-C7C

peptides corresponding to FhuD sequences.........................................................120

Table 3.2. RELIC/MATCH identification of TonB-affinity-selected Ph.D.-12

peptides corresponding to FhuD sequences.........................................................120

Table 3.3. RELIC/MATCH identification of FhuD-affinity-selected Ph.D.-C7C

peptides corresponding to TonB sequences.........................................................123

Table 3.4. RELIC/MATCH identification of FhuD-affinity-selected Ph.D.-12

peptides corresponding to TonB sequences.........................................................123

Table 3.5. DLS analysis of TonB, FhuD and MBP-switch fusion......................126

Page 24: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

xxiv

Table 3.6. Summary of ligand binding parameters fit to a single site saturation

ligand binding model...........................................................................................130

Chapter 4

Table 4.1. Kinetics and affinity of TonB–FhuD interactions according to “1:1

titration” model....................................................................................................158

Page 25: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

Chapter 1

Literature review and thesis objectives

Page 26: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

2

1.0 Role of iron in the bacterial life cycle

This literature review focuses on mechanisms of siderophore-mediated

iron uptake in Gram-negative bacteria, with emphasis placed on the model

organism Escherichia coli. Iron represents a vital element that participates in

many cellular metabolic processes. Most bacteria require iron for growth and

division except for certain lactobacilli species, which replace iron with the

elements manganese and cobalt (1). Cellular processes that require iron include

nucleotide and protein synthesis, respiration, regulation of gene expression,

xenobiotic degradation and maintenance of oxidative homeostasis (2).

Accordingly, iron is a co-factor of many proteins and enzymes including

ribonucleotide reductases, oxidases, cytochromes, peroxidases, and aconitases (2).

Iron’s unique electrochemical properties make it an ideal participant in

processes that require electron transfer. As a first row transition metal, iron

generally exists in one of two oxidation states (3): the ferric (Fe3+

) state and the

ferrous (Fe2+

) state. Under physiological conditions, oxidation states can inter-

convert (3), resulting in an ability to modulate iron’s coordination number. For

this reason, the activities of proteins that utilize iron as a co-factor can be

modulated so as to regulate vital metabolic processes.

1.0.1 Toxicity of iron

Despite iron’s importance as a mediator of cellular function, it also

represents a potentially toxic element due to its propensity to react with oxygen

and reactive oxygen species (ROS). Under aerobic conditions, cellular respiration

Page 27: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

3

produces considerable amounts of ROS such as superoxide and H2O2. Ferrous

iron can react with these species through a series of steps known as Fenton

reactions to generate further ROS such as hydroxyl radicals (4). Through

oxidative mechanisms, hydroxyl radicals damage biological macromolecules such

as proteins, nucleic acids and lipids. For this reason, control of oxidative

homeostasis is vitally important to bacteria. By incorporating into enzymes, iron

acts as a sensor for the cellular redox potential; iron-dependent enzyme activities

become modulated as a function of the co-factor’s oxidation state (3). Through

activities of iron-dependent enzymes, such as superoxde dismutase and catalase,

the toxic effects of ROS are mitigated.

1.0.2 Limitations of iron bioavailability

Despite being the fourth most abundant element on Earth (3), iron is

essentially inaccessible to biological systems. Earth’s aerobic and aqueous

environment stabilize iron’s ferric state (5). Ferric iron combines with hydroxides

to form insoluble ferric hydroxides; the concentration of soluble ferric iron is

reduced to levels below 10-18

M (6). For bacteria that colonize host organisms,

bioavailability is reduced even further by sequestration strategies of the innate

immune system. In this niche, bioavailable iron is present at a concentration of

approximately 10-24

M, a concentration well below the micromolar amounts

required for a single generation of a bacterium’s life cycle (7).

Page 28: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

4

1.0.3 Bacterial iron sources

Bacterial iron sources are governed by the niche that a particular

bacterium occupies. Environmental-dwelling bacteria obtain iron through soil or

water basin sources. Host-colonizing bacteria obtain iron directly from cellular

sources, such as erythrocytes and macrophages, or from protein sources, such as

hemoglobin, transferrin and lactoferrin. Regardless of the source, bacterial iron

acquisition requires passage of the nutrient across biological barriers. For Gram-

negative bacteria, this requires transport of iron across two distinct membranes

that constitute the cell envelope. Since these barriers discourage transport, a

detailed description of the Gram-negative cell envelope is warranted.

1.1 Gram-negative cell envelope

The Gram-negative cell envelope is a structure that is both beneficial and a

liability. Its benefits include functioning as a protective barrier against toxic

compounds, such as solvents, bile salts or antibiotics (8). In addition, the cell

envelope enables attachment to environmental substrates or to host cells, and

enables the bacterium to evade host immune responses. However, the cell

envelope also presents a diffusion barrier that prevents nutrients greater than 700

Da from diffusing into the cytoplasm (9). The composition of the Gram-negative

cell envelope (Figure 1.1) comprises three main components: the outer membrane

(OM), the periplasm (which includes the peptidoglycan layer), and the

cytoplasmic membrane (CM).

Page 29: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

5

1.1.1 Outer membrane

The Gram-negative bacterial OM is an asymmetric structure; exposed to

the extracellular space is a leaflet comprised of lipopolysaccharide (LPS).

Exposed to the periplasmic space is a phospholipid leaflet. Embedded between

these leaflets are proteins that bestow the bacterium with many adaptive

capabilities. Each of these features is now discussed in greater detail.

1.1.2 Lipopolysaccharide

Embedded within the OM outer leaflet are LPS molecules. Each LPS

molecule is a tripartite structure comprising a proximal lipid A moiety, a central

core oligosaccharide and distal O-antigen repeats (10). The lipid A moiety is

essential in forming the integrity of the OM. It possesses phosphorylated

hydrophilic glucosyl-amine head groups and saturated fatty acid tails. Lipid A’s

head groups bestow a net negative charge to the Gram-negative OM, while the

fatty acid tails partition together to form a hydrophobic plane in the outer leaflet.

Connected to lipid A head groups are central, hydrophilic connective core

oligosaccharides, which provide a protective barrier against charged toxins such

as antimicrobial peptides, bile salts and dyes. The central oligosaccharide

comprises two elements: inner and outer cores. The inner core links directly to

lipid A, while the outer core extends into the extracellular space and links to the

distal O-antigen polysaccharides.

Extending beyond the core oligosaccharide into the extracellular medium

are O-antigen repeats. O-antigen repeats are required for virulence, since loss of

Page 30: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

6

these regions often attenuates infectivity (11). These repeats form a polymer of

sugars, which display great variation in chemical composition and number.

Chemical compositions vary depending on a given bacterial species and can

include neutral sugars, amino sugars and uronic acids. In addition, the number of

repeats is also species-dependent. Even within strains of a given species, the

number ranges anywhere from zero to over forty repeats (12).

1.1.3 Inner leaflet of outer membrane

The OM inner leaflet consists of phospholipids. Primarily, inner leaflet

phospholipids are similar in composition to those of the CM, but vary somewhat

depending on bacterial species and strains. In E. coli, the inner leaflet

composition prefers enrichment of phosphatidylethanolamine lipids (13). In

addition, smaller amounts of phosphatidylglycerol and di-phosphatidylglycerol

are found (12).

1.1.4 Outer membrane proteins

In addition to LPS and phospholipid, the OM comprises approximately

50% protein (8). These OM proteins (OMPs) form the basis of selective

permeability, a hallmark of OM function. Diverse properties are ascribed to

OMPs, some of which are described in greater detail later. In general, OMPs are

classified either as porins that allow non-specific diffusion of small solutes into

Page 31: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

7

Figure 1.1. Gram-negative bacterial cell envelope. Illustrated is a schematic of the

Gram-negative bacterial cell envelope. The asymmetric outer membrane comprises two

lipid leaflets: an outer lipopolysaccharide leaflet and an inner phospholipid leaflet. The

remaining OM elements comprise outer membrane proteins (OMPs). OMPs function as

structural proteins (OmpA, Lpp and Pal), or they function as selective permeability

barriers. Porins, such as OmpF, passively uptake water-soluble solutes (preferentially

cations), whereas OMPs, LamB and FadL, facilitate diffusion of maltosaccharides and

long-chain fatty acids, respectively. OMPs such as the transporter, FhuA, actively uptake

ferric-siderophores. Other OMPs such as TolC facilitate efflux. The periplasm is an

aqueous compartment comprised of a peptidoglycan layer and protein. Peptidoglycan is

bound to the OM by OmpA, Lpp and Pal. Periplasmic proteins, such as maltose binding

protein and FhuD, shuttle maltosaccharides and ferric-siderophores, across the periplasm

respectively. The CM is comprised of a phospholipid bi-layer with embedded

transmembrane proteins. CM proteins have major functions such as respiration and

transport. Proteins such as those comprising electron transport complexes actively pump

protons across the CM and generate the PMF. Proteins such as MscL bi-directionally

transport cations across the CM. Active transporters, such as MalFG/K2 and FhuB/C,

hydrolyze cytoplasmic ATP and transport maltosaccharides and ferric-siderophores into

the cytoplasm, respectively. The TonB–ExbB–ExbD complex harnesses energy of the

PMF and transduces it to OM-embedded TonB-dependent transporters such as FhuA.

Other CM proteins such as EntS are responsible for secretion of siderophores from the

cytoplasm to the periplasm.

Page 32: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

8

the periplasm, as enzymes with various catalytic activities, as cell surface

appendages, or as structural elements that maintain OM integrity. OMPs can

traverse both leaflets of the OM as integral membrane proteins or associate with

one leaflet of the OM by a covalently attached lipid as peripheral membrane

proteins (Figure 1.1).

1.1.5 Periplasm

The periplasm occupies space between the OM and CM (Figure 1.1). This

compartment houses key structural components of the cell envelope including the

cell wall (peptidoglycan) and numerous proteins. Proteins found in this

compartment exhibit many diverse functions (discussed in sections 1.1.8 and 1.9).

The volume occupied by this space varies according to environmental factors such

as osmolarity and may be species-specific. Under physiological conditions, the E.

coli periplasm is estimated to have a volume that occupies between 8-20% of total

cell volume and a width between OM and CM ranging from 130 to 250 Å (14).

1.1.6 Peptidoglycan

The cell wall structure provides a rigidity that gives bacteria their

characteristic shapes (Figure 1.1). It also prevents cytoplasmic contents from

rupturing under conditions of low osmolality (15). Structurally, peptidoglycan is

a heteropolymer comprising roughly equal amounts of polysaccharides that are

linked to peptides. The basic repeating peptidoglycan unit is shared amongst most

Gram-negative bacteria and comprises two linked N-acetylglucosamine (NAG)

Page 33: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

9

and N-acetylmuramic acid (NAM) sugar residues that are derivatized with

oligopeptides (16). Each NAG-NAM repeat is linked continuously to another

NAG-NAM repeat to form a linear backbone. Projecting away from this

backbone are oligopeptides, which form cross-links with adjacent peptidoglycan

strands to yield the peptidoglycan scaffold.

The peptidoglycan scaffold is a vast sieve-like three-dimensional structure

that encompasses the bacterium. An NMR structure of a NAG-NAM

pentapeptide revealed the structural basis of these properties (17). From the

pentapeptide structure, a three-dimensional model was built; NAG-NAM residues

formed a linear backbone from which the pentapeptide linkers extended. Sieve-

like characteristics were formed by pores between the model’s adjacent cross-

linked peptidoglycan strands. Pore diameters were estimated from this model and

ranged from 70 to 100 Å, wide enough to accommodate passage of nutrients and

even proteins.

The orientation of pore openings with respect to the OM plane is a debated

subject. Currently, two conflicting reports have provided evidence for the

direction of pore orientations. While the NMR study suggested that pores orient

perpendicular to the OM plane (17), an electron cryotomography study suggested

parallel orientations (18). Despite these differences, perpendicular orientations

are attractive since the pore diameters are large enough to accommodate proteins,

especially those either targeted to the OM or those with OM-proximal activities.

Page 34: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

10

1.1.7 Lipoproteins

The peptidoglycan layer is secured to the OM by interactions with various

proteins. Three well-characterized E. coli peptidoglycan binding proteins include

OmpA, lipoprotein (Lpp) and Pal (Figure 1.1). OmpA is a ubiquitous structural

OMP that possesses two domains. Its N-terminal transmembrane domain folds

into a monomeric, 8-stranded β-barrel, while its C-terminal periplasmic domain

folds into a peptidoglycan-binding motif (19). Lpp is the most abundant protein

in E. coli with an estimated 700,000 copies per bacterium (20). It forms a

trimeric, coiled-coil structure that bridges its N- and C-termini (21). It

intercalates within the OM inner leaflet by three lipids covalently attached to its

N-terminus, and covalently attaches to peptidoglycan by means of its C-terminal

lysine residue (22). Like OmpA and Lpp, Pal attaches to the inner leaflet of the

OM by its N-terminus, while its C-terminus interacts with peptidoglycan (23).

Together, OmpA, Lpp, and Pal secure the peptidoglycan layer in close proximity

to the OM inner leaflet.

1.1.8 Periplasmic proteins

Many proteins exist within the periplasm. Some are enzymes, while

others bind nutrients (Figure 1.1). In addition, some OM- and CM-embedded

proteins possess significant periplasmic domains. Periplasmic enzymes regulate a

large variety of physiological processes including OM biogenesis, peptidoglycan

assembly, protein folding, disulfide formation, surface appendage assembly and

protein secretion. Periplasmic nutrient-binding proteins represent a large and

Page 35: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

11

diverse class of proteins and are discussed in section 1.9. The abundance of

periplasmic proteins, taken together with a limited periplasmic volume and

peptidoglycan sieving properties, suggests that the periplasm is a highly viscous

and gel-like compartment (14). This property results in drastically reduced

diffusion-facilitated nutrient transport, an important theme in the context of

bacterial iron acquisition.

1.1.9 Cytoplasmic membrane

The CM separates cytoplasmic contents from the rest of the bacterium. It

comprises roughly equal amounts of phospholipid and protein content.

Phospholipid compositions vary between species, but generally comprises 70-

80% phosphatidylethanolamine, 15-25 % phosphatidylglycerol and 5-10%

cardiolipin (24). In addition, small amounts of metabolic by-products can

incorporate into the membrane. Many proteins embed within the CM and

participate in physiological processes including cell wall synthesis, protein and

small molecule secretion, nutrient uptake and respiration, of which the latter

activity generates CM-associated proton motive force (Figure 1.1).

1.2 Nutrient transport across the cell envelope

The OM of Gram-negative bacteria provides an efficient barrier against

toxic compounds. However, it also prevents diffusion of larger nutrients,

including iron in the form most often acquired by bacteria. Therefore, bacteria

must utilize a limited number of mechanisms for passage of nutrients across the

Page 36: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

12

OM. Mechanisms include passive transport, facilitated transport, and active

transport. Each mechanism is now discussed in greater detail.

1.2.1 Passive transport

Passive diffusion is the simplest nutrient uptake mechanism, whereby

small hydrophilic solutes diffuse non-specifically through OM porins. Diffusion

is permitted by differential permeabilities afforded by porin transmembrane

channels. Examples include the cation-selective porins OmpC and OmpF and the

anion-selective porin PhoE. Structural data (Figure 1.2) revealed how porins

permit non-specific diffusion; trimeric β-barrel assemblies insert into and traverse

the OM (25-27). Under physiological conditions, barrel diameters support

passive diffusion of solutes smaller than 700 Da. Diffusion through these pores is

driven by concentration gradients; when concentrations are high enough, nutrients

non-specifically diffuse against their concentration gradient into the periplasm.

Nutrients are then transported across the CM by channels such as the

mechanosensitive channel, MscL (28) (Figure 1.1).

1.2.2 Facilitated transport

Facilitated diffusion is a second mechanism of porin-mediated nutrient

transport. Less concentrated and rare nutrients that cannot diffuse against a

concentration gradient are transported by this mechanism. The maltosaccharide

transporter, LamB, is a canonical facilitated diffusion porin.

Page 37: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

13

Figure 1.2. E. coli OM nutrient transporters. Structures are displayed as ribbon representations

and viewed laterally from the plane of the OM (top row) or viewed from the extracellular space

into the transporter lumens (bottom row). For clarity, monomeric lateral views are displayed for

OmpF and LamB. Where present, transporter ligands are displayed as surface representations and

metals are displayed as grey spheres. Left: Passive cation transporter OmpF (PDB code 2J1N);

Middle: facilitated-diffusion maltosaccharide transporter LamB (PDB code 1MPM); Right: active

ferric-siderophore transporter FhuA (PDB code 1QFF).

LamB folds into an 18-stranded β-barrel channel (29) that traverses the OM and,

like OmpF, forms trimers (Figure 1.2). A high affinity sugar binding site is

created by three extracellular loops that fold into and constrict LamB’s lumen

(30). Sugars, such as maltose, bind within this site and are funnelled further into a

lumenal arrangement of residues that facilitate diffusion into the periplasm.

Maltose is then bound by periplasmic maltose-binding protein and delivered to

CM-embedded permease MalFG/K2 (Figure 1.1). ATP hydrolysis facilitates

maltose translocation through MalFG/K2 and into the cytoplasm.

Another OMP that facilitates nutrient diffusion is the OM long chain fatty

acid receptor, FadL. Like the porins described above, FadL forms a

transmembrane β-barrel, which possesses an extracellular, high affinity binding

Page 38: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

14

site for long chain fatty acids. However, FadL embeds within the OM as a

monomer. Diffusion of fatty acids into the OM is postulated to occur through a

channel within FadL that extends into the barrel lumen. A portal along one side

of the barrel, and which connects to the lumenal channel, is the postulated access

route to the OM outer leaflet (31). Unlike ions and maltosaccharides, the fate of

fatty acids transported through FadL is less known. Fatty acids might

spontaneously diffuse across the cytoplasm or bind to an unidentified binding

protein. However, little evidence for these mechanisms has been reported.

1.2.3 Active transport

Mechanisms of passive and facilitated diffusion enable uptake of small

nutrients. To facilitate transport of nutrients larger than 700 Da, such as iron

bound to siderophores (discussed in section 1.4), active transport mechanisms are

required. The best-characterized active transporters include CM-embedded

permeases, such as MalFG/K2, which couple active transport to ATP hydrolysis.

However, there is no energy source available to OM transporters. Bacteria have

overcome this limitation by transducing CM-derived energy across the cell

envelope and to the OM. The CM-embedded TonB–ExbB–ExbD multi-protein

complex (Figure 1.1) harnesses and stores energy from the proton motive force

(PMF) and transduces this energy to TonB-dependent iron transporters found in

the OM, such as the ferric-hydroxamate receptor FhuA (Figure 1.2). Mechanisms

of active transport are now discussed in greater detail.

Page 39: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

15

1.3 General bacterial strategies for iron acquisition

Iron acquisition is a bacterial paradox. Most require it for survival, yet it

is present in miniscule amounts and its acquisition requires passage through

barriers generated by the cell envelope. To overcome this limitation, bacteria

have evolved specialized iron transport systems. The specific system employed

by a particular bacterium depends on the oxidative state of iron found within that

bacterium’s environment; specialized transport systems exist for uptake of ferrous

iron and for uptake of ferric iron.

1.3.1 Ferrous iron transport

Bacteria that occupy anaerobic or micro-aerobic niches such as the

gastrointestinal tract can obtain soluble ferrous iron by ionic transport systems.

The best-characterized ferrous ion uptake system is the E. coli Feo system, which

is utilized to colonize the gastrointestinal tract (32). A detailed understanding of

how this system operates has yet to be realized. Poorly understood factors include

the mechanisms of ferrous iron transport across the OM and periplasm.

Presumably, ferrous iron translocates into the periplasm through an unidentified

OM porin. An unidentified periplasmic binding protein may then transport

ferrous iron to the CM.

Better understood is the mechanism of ferrous iron transport across the

CM. FeoB, a CM-embedded protein is postulated to actively transport ferrous

ions into the cytoplasm (33). However, unlike the previously described CM

permeases, FeoB displays activity only when coupled to GTP hydrolysis. Other

Page 40: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

16

proteins including FeoA and FeoC may facilitate FeoB-mediated transport, yet

their exact roles require further investigation.

1.3.2 Ferric iron transport

Bacteria that obtain ferric iron have evolved different acquisition

strategies. One strategy is to directly acquire iron from host iron-binding proteins.

By capturing proteins such as transferrin, lactoferrin and hemoglobin, bacteria can

exploit rich sources of iron. Specialized TonB-dependent transporters (TBDTs),

such as the Neisseria meningitidis and Neisseria gonorrhoeae transferrin receptor,

TbpA, bind transferrin at the bacterial surface and uptake ferric iron after

stripping it from transferrin (34,35). In addition, a lactoferrin receptor from

Helicobacter pylori has been described (36) that binds ferric-lactoferrin and

uptakes the ferric iron, presumably by a mechanism similar to TbpA.

Synthesis and extracellular secretion of hemophore proteins is a second

bacterial ferric iron acquisition strategy. Hemophores, such as HxuA from

Haemophilus influenza, and HasA from various bacteria including Serratia

marcescens, Pseudomonas aeruginosa, and Yersinia pestis (37) bind to and strip

heme from host proteins hemopexin and hemoglobin, respectively. Having

stripped heme, HxuA and HasA present it to cognate bacterial surface TBDTs.

Heme is subsequently released from hemophore and transported through the

TBDT into the periplasm.

Bacteria also synthesize and secrete siderophores into their extracellular

environment. Siderophores represent a large and diverse class of small organic

Page 41: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

17

molecules that possess extremely high affinities for ferric iron, allowing them to

strip and bind iron from minerals and from host iron-binding proteins. Bacterial

ferric-siderophore transport is the focus of this thesis; having bound iron, ferric-

siderophores are transported back into the bacterium using mechanisms described

in the remainder of this review. In general, transport (Figure 1.1) entails capture

of ferric-siderophore by an OM siderophore transporter, such as the ferric-

hydroxamate receptor, FhuA (discussed in section 1.5). By a TonB-dependent

mechanism, siderophores are then actively transported through the receptor and

into the periplasm. Periplasmic binding proteins such as the ferric-hydroxamate-

binding protein, FhuD, then bind the siderophore and deliver it to a CM-

embedded permease, such as the ferric-hydroxamate permease FhuB/C. Given

the diversity of siderophores produced and employed during bacterial iron

acquisition, their properties are now discussed in greater detail.

1.4 Siderophores

Siderophores represent a diverse class of small organic molecules (Mr < 1

kDa) that bind iron with extremely high affinity. Affinities between siderophore

and ferric iron vary; association constants (Ka) have been reported between 1022

to

1052

(38,39). Siderophores are synthesized and secreted by bacteria and fungi

alike and help in the colonization of their environments. Given the importance of

solubilizing and chelating iron for colonization, the abilities to produce and utilize

siderophores are known as virulence factors for many pathogenic bacteria. Over

three hundred siderophores have been characterized, and are classified by the

Page 42: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

18

chemistries used to chelate ferric iron. Despite the diversity of scaffolds that

comprise siderophore backbones, most chelate iron with bidentate oxygen

functional groups. Most siderophores belong to one of four different classes:

catecholates (including phenolates), hydroxamates, α-hydroxycarboxylates and

mixed-types (40).

Perhaps the best-characterized siderophore is the catecholate, enterobactin,

the only endogenous siderophore produced by E. coli K-12. The enterobactin

backbone comprises three linked L-serine residues, each derivatized with iron-

chelating di-hydroxybenzoic acid groups. The three serine residues incorporate

into a tri-lactone macrocycle to form a symmetrical scaffold (Figure 1.3).

Phenolate siderophores are similar to catecholates, except for use of a mono-

hydroxybenzoic acid functional group. Examples of phenolate siderophores

include yersiniabactin from Y. pestis and pyochelin from P. aeruginosa.

Produced exclusively by fungi, the hydroxamate-type siderophore

ferrichrome, represents one of the first-characterized microbial iron chelator (6).

Structural scaffolds of hydroxamate siderophores consist of two tripeptides: a

single tripeptide (containing glycine, alanine or serine) linked to a second

tripeptide of derivatized L-ornithine functional groups (Figure 1.3). Iron

chelation occurs within the hydroxamate groups of derivatized ornithines.

Carboxylates represent the third class of siderophores used to chelate iron.

Chelation from this class of siderophores occurs from α-hydroxycarboxylic acid

functional groups. Examples include staphyloferrin A from Staphylococcus

species and achromobactin from Erwinia chrysanthemi. In addition, citrate, a

Page 43: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

19

metabolic intermediate, efficiently chelates iron and is transported into bacteria

(Figure 1.3).

Siderophores also incorporate mixed functional groups into their scaffold.

Many mixed siderophores have been described and include such combinations as

citrate-hydroxamate functional groups in aerobactin (Figure 1.3), citrate-

catecholate functional groups in petribactin and catecholate-hydroxamate in

heterobactin B.

Figure 1.3. Siderophores transported by E. coli. A. The catecholate siderophore, enterobactin.

Enterobactin is the only endogenous siderophore produced by E. coli K-12; B. the hydroxamate

siderophore, ferrichrome. R-groups represent sites of species-specific, variable chemical

modifications; C. the carboxylate, citrate. Citrate is a naturally occurring metabolic by-product;

D. the mixed-type siderophore, aerobactin. Aerobactin chelates iron using a combination of

hydroxamate and carboxylate chemistries. Images are adapted from reference 6.

Page 44: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

20

1.4.1 Siderophore biosynthesis

Siderophores are synthesized from assemblies of cytoplasmic enzymes

that create bonds using non-ribosomal peptide biosynthesis. Modular enzymes

comprise the siderophore biosynthetic machinery; separate domains catalyze step-

wise assembly of siderophores without need for an RNA template (38). Genes

that encode siderophore biosynthetic enzymes localize in operons with

siderophore-specific locations. For example, in E. coli, genes for enterobactin

biosynthesis cluster within an operon at a single chromosomal locus shared by

genes encoding enterobactin uptake proteins (38). In contrast, genes for synthesis

of vibriobactin from Vibrio cholerae localize within one of its two chromosomes,

but stagger within two different gene clusters, separated by nearly 100 bp (38).

Other bacteria such as the fish pathogen Vibrio anguillarum harbour virulence

plasmids that encode genes for siderophore biosynthesis, in addition to the

cognate siderophore uptake proteins (38).

1.4.2 Siderophore secretion

Once synthesized, siderophores are secreted into the extracellular

environment. Compared to fungi, less is known about secretion of bacterial

siderophores. Enterobactin secretion by E. coli has been studied. The CM-

embedded protein EntS is known to translocate enterobactin across the CM into

the periplasm (40). EntS belongs to the major facilitator superfamily of proteins

that exhibit a broad range of export activities. Once in the periplasm, enterobactin

is postulated (40) to translocate to the extracellular space by transport through

Page 45: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

21

OM channel TolC (Figure 1.1). Having translocated through TolC and scavenged

iron with high affinity, ferric siderophores are then transported back into the

bacterium by various TBDTs, discussed below.

1.5 TonB-dependent transporters

Many TBDTs have been discovered. As high affinity receptors, each

binds and transports specific siderophores. TBDTs expressed in E. coli include

the ferric-hydroxamate receptor FhuA, the ferric-enterobactin receptor FepA, and

the ferric-citrate receptor FecA. In addition, TBDTs also transport rare metallo-

nutrients such as the E. coli vitamin B12 (cobalamin) receptor BtuB. Many

TBDTs are also exploited as receptors for entry of various phage and antibacterial

compounds. The remainder of this section focuses on structural knowledge

gained from TBDTs with emphasis placed on the E. coli transporter FhuA.

1.5.1 Structures of TonB-dependent transporters

FhuA is receptor for ferric hydroxamate-type siderophores such as

ferrichrome. In addition, it serves as receptor for bacteriophages T1, T5, Φ80,

UC-1, and the antibacterial compounds colicin M, microcin-25, albomycin and

rifamycin CGP 4832 (41). FhuA is a 79 kDa protein possessing 714 residues that

fold into two unique domains. Residues 1–160 form a globular cork domain that

inserts into a C-terminal, 22-stranded β-barrel domain comprised of residues 161–

714. FhuA was the first TBDT structure (1998) to emerge at atomic-resolution

Page 46: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

22

detail (Figures 1.2 and 1.4) and revealed a canonical fold that is conserved

amongst all TBDTs with known structure (42).

FhuA’s structure revealed many striking features. First, unlike smaller

porins that form trimers in the OM, the structure of FhuA revealed a monomer.

Bound to FhuA was a single LPS molecule that indicated how this class of lipids

can interface and surround the outer circumference of an OMP (43). The most

striking observation revealed that FhuA’s globular cork, comprising a four-

stranded β-sheet surrounded by four short α-helices, fits tightly within the barrel

domain (Figure 1.2). No obvious channel was observed within the cork and

barrel, highlighting the need for structural rearrangement in order for siderophore

transport to occur.

Structures of many E. coli TBDTs have since been determined (Figure

1.4), including those of FepA (44), FecA (45), BtuB (46-49), and the Colicin Ia

receptor Cir (50). Structures of TBDTs from P. aeruginosa have also been

solved (Figure 1.4), including the ferric-pyochelin transporter FptA (51) and the

ferric-pyoverdine transporter FpvA (52). Most recently, TBDT structures have

been solved from other organisms including the hemophore receptor HasR (53)

from S. marcescens and the ferric-alcaligin transporter FauA (54) from

Bordetella pertussis (Figure 1.4).

Structural features of TBDTs are similar. All share architectures

comprised of cork domains that fold and insert into β-barrel domains. The β-

barrel domains nearly superimpose; connecting subsequent barrel strands are short

periplasmic turns and longer extracellular loops. Siderophores bind within sites

Page 47: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

23

Figure 1.4. Structures of TonB-dependent transporters. Displayed are ribbon representations of

TBDTs. Cork domains are coloured separately from barrel domains. Ligands are represented as

surface models where present.

comprised of each receptor’s extracellular loops and cork apices. Structural

differences arise mainly from the lengths of extracellular loops and from

specialized siderophore binding sites. In all structures, the corks prevent passive-

or facilitated-diffusion of siderophore through receptor lumens. This necessitates

active transport by interaction with TonB; interactions between TonB and TBDTs

facilitate opening of a lumenal pore large enough to allow siderophore

translocation.

1.5.2 Ton boxes

All TBDTs share a common N-terminal feature called the “Ton box” that

functions as a molecular recognition motif for TonB. TonB initially interacts with

siderophore bound-receptors through this motif (discussed in greater detail later).

Page 48: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

24

Though not strictly conserved, Ton boxes generally comprise the consensus:

D/ETX1X

2VX

3A, where X

1 and X

2 are hydrophobic amino acids and X

3 is any

amino acid (55). Ton boxes are unresolved in most TBDT structures, but were

resolved as extended conformations in the structures of FepA, BtuB and CirA.

Noteworthy, is that the conformation of BtuB’s Ton box differed when

crystallized in a lipid cubic phase, highlighting both the dynamic nature of the

Ton box, as well as its conformational dependence on chemical environment, a

point re-visited below.

1.5.3 Transcriptional regulatory domains

Transporters such as FecA and FpvA have additional extensions N-

terminal to their Ton boxes that function as transcriptional signalling domains.

Through mechanisms not completely understood, the signalling domains help

regulate transcription of genes in the transporter biosynthetic operon. Structures

of isolated FecA signalling domains have been solved by NMR (56,57) and of the

FpvA signalling domain bound to the transporter by X-ray crystallography (58).

The structures revealed nearly identical folds, comprising two antiparallel β-

sheets flanked by alternating α-helices. The structure of FpvA with its intact

signalling domain (Figure 1.4) exhibited unresolved space near the receptor’s Ton

box. This was interpreted to mean that space occupied by a transporter’s

signalling domain should not spatially occlude a region that TonB is known to

bind.

Page 49: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

25

1.5.4 Siderophore-induced conformational changes

The structure of FhuA revealed substantial conformational changes that

occur upon binding the ferric-hydroxamate siderophore, ferrichrome.

Ferrichrome binds to an extracellular surface, within a hydrophobic pocket

formed by FhuA’s three cork apices. In addition, extracellular loop 3 and β-

strands 7 and 9 contribute to the binding site. Ferrichrome makes various

hydrogen bond and van der Waals contacts with conserved FhuA residues.

Binding is strong, with a measured KD of around 200 nM (42).

Upon binding ferrichrome, FhuA exhibits conformational changes that

propagate to the receptor’s periplasmic surface. While the barrel conformation

only slightly changes (RMSD 0.4 Å between apo- and ferrichrome-bound FhuA),

the cork undergoes significant rearrangement. Compared to apo-FhuA, cork

apices translocate approximately 2 Å towards the iron atom when ferrichrome is

bound. The most striking conformational changes localize to FhuA’s periplasmic

face. Upon binding ferrichrome, residues 24–29 (termed the switch helix) extend

and translocate away from a helix-stabilizing hydrophobic groove (Figure 1.5).

Translocation is considerably large; residue Trp-22 displaces approximately 17 Å

from its location in the apo-FhuA state. This structural transition is postulated to

signal a state of ligand occupancy that extends into the periplasm, whereby TonB-

dependent energy transduction begins. However, it is unclear whether this

transition is a crystallization artefact. It has been reported that crystallization

solutes can alter conformations of membrane proteins (59). Furthermore, an

Page 50: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

26

Figure 1.5. FhuA exhibits periplasmic conformational changes upon binding ferrichrome.

Detailed ribbon representations of apo-FhuA (PDB code 1QFG, left) and ferrichrome-bound FhuA

(PDB code 1QFF, right) are displayed as viewed from the periplasm, looking into FhuA’s lumen.

FhuA structural elements are coloured as follows: β-barrel domain (blue), cork domain (orange),

N-terminal switch helix residues (green). Ferrichrome is displayed as a red surface representation.

For clarity, extracellular loops are not shown.

electroparamagnetic resonance spectroscopy (EPR) study indicated that in

solution, both apo- and ferrichrome-bound FhuA’s switch helix remains unwound

and in an extended conformation (60).

Shortly after the ferrichrome-bound structure, additional FhuA structures

with various bound ligands were solved. These included the natural siderophore-

antibiotic, albomycin-bound structure (61), the siderophore, phenylferricrocin-

bound structure (61), and the antibiotic-siderophore conjugate, rifamycin CGP

4832-bound structure (62). All ligands occupied regions proximal to the

ferrichrome binding site. Slight differences were observed between binding

modes for the different ligands.

As with ferrichrome, albomycin and phenylferricrocin both elicited the

same periplasmic conformational change, whereby FhuA’s switch helix unwound

and translocated nearly 17 Å away from its original position. However, due to a

slightly different binding mode of rifamycin CGP 4832, the switch helix

Page 51: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

27

conformational change was not observed; its binding does not affect the position

of FhuA’s cork apex A. In contrast, binding of the other ligands causes upward

movement of apex A, which propagates distally to the periplasmic face and causes

unwinding of FhuA’s switch helix.

Structures of other ligand-bound TBDTs from E. coli indicated that

binding mechanisms are not strictly conserved. Some similarities to FhuA were

apparent; binding determinants generally involve use of cork apices and

extracellular loops. However, each receptor tends to use a different combination

to fulfill this purpose. In addition, ligand-induced conformational changes differ

slightly for each transporter. For example, the structure of ferric-enterobactin

transporter, FepA, in its apo-state, failed to provide evidence for a switch helix

conformational change (44). The significance of this outcome is not apparent;

bound enterobactin was never resolved, despite having soaked FepA crystals with

the ligand. Assuming that enterobactin at least partially occupied the crystal

lattice, it was concluded that unwinding of a receptor’s switch helix is not a

conserved mechanism for signalling ligand occupancy. Despite uncertainties

associated with the FepA structure, its Ton box was resolved and provided first

evidence that the motif tends to adopt extended and loosely structured

conformations.

Structures of the ferric citrate-bound transporter, FecA, emulate the

ligand-binding features exhibited by FhuA; three cork apices and different

combinations of extracellular loops bind ferric-citrate (45,63). In contrast, FecA’s

siderophore binding site is hydrophilic and unlike FhuA, FecA’s extracellular

Page 52: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

28

loops 7 and 8 exhibit large conformational changes upon binding ferric-citrate that

close over the ligand binding site, rendering it solvent-inaccessible. Like FhuA,

apo-FecA possesses an N-terminal switch helix that partially unwinds upon

binding ferric-citrate, suggesting similar ligand-induced signalling mechanisms.

Further supporting this mechanism was the observation that FecA’s Ton box,

partially resolved and extended in the apo-structure, undergoes a structure-to-

disorder transition upon binding ferric citrate. Presumably, the disordered Ton

box is flexible and further extended towards the periplasm where it can interact

with TonB.

Structures of TonB-dependent cobalamin transporter, BtuB, with various

ligands bound revealed further differences between ligand binding modes of the

various TBDTs. A structure of cobalamin-bound BtuB demonstrated that cork

apices and five extracellular loops contact the ligand (46). Compared to apo-

BtuB, cork residues 85–96 shift upward by nearly 6 Å towards bound cobalamin.

BtuB also serves as receptor for colicin E2. A BtuB structure in the presence of

colicin E2’s BtuB-binding domain demonstrated remarkably similar binding

modes between BtuB and the colicin E2-derived ligand as compared to BtuB and

cobalamin (49). Main differences include residues contacting the colicin E2-

derived ligand and induced ordering of loops 5 and 6, which are disordered in

apo- and cobalamin-bound BtuB. BtuB’s mechanism of ligand-induced signalling

is also likely different since, unlike FhuA, FepA and FecA, residues with a switch

helix-like conformation were not present in BtuB. However, the order-to-disorder

transition of ligand signalling may be conserved; upon binding cobalamin, BtuB’s

Page 53: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

29

Ton box undergoes a rotation of nearly 180° and becomes slightly more

disordered.

Despite modest differences between ligand binding modes among TBDTs,

all exhibit ligand-induced alterations proximal to their Ton boxes. These

structural alterations are postulated to function as a periplasmic signal of ligand

occupancy that initiates transport. Further support for this mechanism derives

from EPR studies of various TBDTs. When coupled to EPR spectroscopy, the

technique of site-directed spin labelling allows direct measurements of protein

structural dynamics in solution. This approach confirmed that Ton boxes of FecA

and BtuB undergo order-to-disorder transitions upon ligand binding (60).

Unexpectedly, these methods demonstrated that FhuA’s Ton box is always

disordered and that the switch helix conformation observed in crystal structures

may be an artefact of the crystallization conditions. A second EPR study

demonstrated that, in addition to the cobalamin-induced order-to-disorder

transition, ligand binding promotes extension of BtuB’s Ton box by nearly 30 Å

into the periplasm (64). Despite different conclusions between crystallographic

and biophysical studies, all suggest that ligand binding within extracellular loops

of TBDTs propagates a periplasmic signal for recruitment of TonB.

1.6 The TonB–ExbB–ExbD complex

Ligand-bound TBDT structures failed to reveal obvious lumenal

translocation pathways; translocation requires energy input. The CM-embedded

TonB–ExbB–ExbD complex transduces energy to ligand-bound TBDTs. By

Page 54: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

30

harnessing chemiosmotic potential of the CM-derived PMF, the TonB–ExbB–

ExbD complex conformationally energizes TonB so that it can transduce stored

energy to ligand-bound TBDTs. The proteins and interactions that comprise the

TonB–ExbB–ExbD complex (Figure 1.6) are now examined in greater detail.

1.6.1 TonB: Energy transducer

TonB transduces stored energy to ligand-bound TBDTs. Originally, TonB

was thought to provide energy solely for iron and cobalt (via cobalamin) uptake.

However, novel TonB-dependent activities have been reported including uptake

of nutrients such as Ni, sucrose and sulphate (65-67). Properties of TonB vary

greatly between bacteria and include differences in length and the number of

expressed isoforms (68). TonB from E. coli is a 239-residue protein with a

molecular weight of approximately 25 kDa (Figure 1.6). It contains three

domains: a single 32-residue N-terminal transmembrane (TM) domain, a central

proline-rich region and a structured C-terminal domain. TonB interacts with

ExbB and ExbD through its TM helix. The remainder is periplasmic. Residues

33 to around 150 are primarily unstructured and include the central proline-rich

domain (residues 66–100) consisting of alternating Lys-Pro, Glu-Pro repeats.

TonB’s C-terminal domain forms a structured region that directly interacts with

TBDTs. These elements are now examined in greater detail.

Page 55: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

31

Figure 1.6. The TonB–ExbB–ExbD complex. Schematic representation of the TonB–ExbB–

ExbD complex is illustrated. Transmembrane α-helical domains are represented by cylinders.

Protein N- and C-termini are labelled accordingly. Structured regions of TonB’s and ExbD’s

periplasmic C-terminal domains are represented by ovals. TonB’s central, periplasmic proline-

rich region, is represented by a series of curved blue boxes. Directional flow of protons through

the complex is highlighted in red. Periplasmic and cytoplasmic compartments are labelled

accordingly. Proteins are not drawn to scale. For clarity, only one of each protein is displayed.

1.6.2 TonB proline-rich region

Residues 66–100 of E. coli TonB form a central proline-rich region.

Synthetic oligopeptides corresponding to proline-rich regions from E. coli and S.

typhimurium were characterized by NMR spectroscopy (69,70). Both peptides

were found to be elongated and rigid in solution and were postulated to function

as a linker enabling TonB to interact with ligand-bound OM transporters.

However, the exact role of this region is speculative; in some bacteria the

corresponding region is not as proline-rich as E. coli TonB, and in other species

the region can include up to 283 residues of Pro repeats (68). Furthermore, the

periplasm

cytoplasm

Page 56: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

32

region can be deleted without greatly affecting TonB activity in vivo (71). As

discussed below, some studies attribute properties to the region that affect

interactions between TonB and its protein binding partners.

1.6.3 TonB C-terminal regions form a compact structure

C-terminal residues of TonB form a structured domain that is essential for

interactions with OM transporters. Crystal structures of various E. coli

periplasmic TonB derivatives have been crystallized and revealed unique, length-

dependent properties (Figure 1.7). The first C-terminal TonB structures of

derivatives possessing residues 155–239 and residues 162–239 revealed identical,

tightly intertwined dimers (72,73). Compared to shorter derivatives, a slightly

longer derivative that possessed residues 148–239 also crystallized as a dimer, but

much more loosely associated (74). By NMR, an even longer derivative that

possessed residues 103–239 was monomeric in solution (75). In agreement, a

similar TonB derivative from the fish pathogen V. anguilerum that possessed

residues 121–206 was also monomeric by NMR (76).

1.6.4 Oligomeric state of TonB

The in vitro and in vivo oligomeric states of TonB have been thoroughly

investigated. The TonB crystal structures of various periplasmic derivatives

demonstrated a length-dependent tendency to dimerize. Analytical

Page 57: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

33

Figure 1.7. Structures of C-terminal, E. coli TonB derivatives. Structures are aligned according to

the NMR structural orientation (right) and displayed as ribbon representations. Dimeric TonB

structures are coloured as follows: Chain A (blue) and Chain B (green) Left: TonB residues 155–

239 form tightly intertwined dimers (PDB code 1IHR); Middle: TonB residues 148–239 form

loosely associated dimers (PDB code 1UO7); Right: TonB residues 152 -239 forms a monomer

(PDB code 1XX3).

ultracentrifugation (AUC) studies later confirmed these findings; derivatives of

TonB that possess residues 153–239 and fewer sediment as dimers, whereas TonB

derivatives possessing residues 143–239 and longer sediment as monomers

(73,77). The findings that longer TonB derivatives are monomeric in vitro,

suggests that in vivo TonB is also monomeric. However, one study that fused the

cytoplasmic domain of ToxR to TonB’s N-terminus concluded that TonB is

dimeric in vivo (78). The TonB fusions supported TonB-dependent functions and

promoted transcription of the cholera gene, an outcome possible only if the ToxR

domains formed dimers (and by analogy, TonB formed dimers). A second study

confirmed this finding by mutating selected aromatic residues in TonB’s C-

terminus to cysteine; TonB dimers spontaneously formed in a PMF-dependent

manner (79). These conflicting outcomes between in vitro and in vivo

characterizations have yet to be reconciled.

Page 58: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

34

1.7 Protein–protein interactions involving TonB

Interactions between TonB and its E. coli protein partners are now

described in three parts. Interactions between TonB and ExbB/ExbD are

discussed first. Second, interactions between TonB and TBDTs, a subject of

intense investigation, are discussed in greater detail. Finally, interactions between

TonB and other cell envelope proteins with no obvious physiological roles are

described.

1.7.1 TonB–ExbB–ExbD interactions

Interactions between TonB and CM-embedded ExbB and ExbD proteins

are required for energy transduction across the cell envelope. TonB, ExbB and

ExbD are all membrane proteins that possess varying numbers of TM helices.

Compared to TonB, less is known about the ExbB and ExbD proteins. Both are

TM proteins with contrasting properties. ExbB from E. coli is a 24 kDa protein

comprised of 244 residues that form three TM α-helices connected by loops of

varying length (Figure 1.6). ExbB’s longest loop, which connects TM helices 1

and 2, was demonstrated to project into the cytoplasm (80) where it forms the

bulk of ExbB’s non-membrane-embedded composition.

ExbD is a smaller 16 kDa protein comprised of 141 residues that form a

single TM α-helix. The bulk of ExbD extends into the periplasm (Figure 1.6). A

structure of ExbD’s periplasmic domain from E. coli was solved by NMR (81)

and revealed a highly flexible protein that exhibited structure only at low pH.

Page 59: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

35

Unexpectedly, the fold of ExbD’s periplasmic domain was similar to structural

elements of periplasmic siderophore binding proteins FhuD and CeuE.

Together, TonB, ExbB and ExbD form a complex that harnesses the

chemiosmotic potential of the PMF. The TonB–ExbB–ExbD complex must

therefore generate TM channels that facilitate proton translocation. This large

multi-protein assembly is postulated to oligomerize into a complex with a

stoichiometry of 1:7:2 (TonB:ExbB:ExbD) (82). However, aside from this

postulated stoichiometry, less is known about how the proteins assemble in the

CM. A computational model for TM assembly of the complex has been reported

(83). Sequence and domain conservation between ExbB/D and MotA/B proteins

provided a foundation for development of a homologous TonB–ExbB–ExbD

complex. MotA and MotB form a CM-embedded complex that harnesses PMF to

drive rotation of bacterial flagella. Models that matched TM components of

MotA/B with TM components of TonB–ExbB–ExbD enabled TM domain

alignment such that a plausible proton channel was visualized. Importantly,

ionizable residues within the model’s channel and that could promote proton

translocation include conserved ExbB residues. When mutated, these residues

have demonstrable effects on TonB-dependent transport (84). In addition,

mutations of conserved and ionizable TonB residues that line the putative channel

also affect TonB-dependent transport (85,86).

Interactions between TonB and ExbD were recently characterized in vivo.

Both protiens formed PMF-dependent and periplasm-localized, formaldehyde

cross-linked heterodimers, indicating that TonB–ExbD interactions are not solely

Page 60: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

36

localized to their TM domains (87). Periplasm-localized regions of ExbD also

formed cross-linked homodimers. However, unlike formation of TonB–ExbD

heterodimers, ExbD homodimers formed, independent of PMF.

1.7.2 TonB–OM receptor interactions

TonB interacts with TBDTs within at least two distinct localizations: Ton

box regions and cork/β-barrel regions. TonB–FhuA interactions were originally

demonstrated in vivo by complementary suppressor mutations between FhuA and

TonB (88); interactions localized binding of TonB to FhuA’s Ton box. Later

studies using formaldehyde cross-linking and biochemical assays confirmed that

TonB bound to FhuA in vitro and to FepA in vivo (89,90) and localized binding of

TonB to FepA’s Ton box (91). In addition, TonB–BtuB Ton box interactions

were extensively studied. By site-directed, disulfide cross-linking and

mutagenesis, the Ton box of BtuB was demonstrated to interact with TonB around

TonB residue 160 in a conformation-dependent manner (92,93). Most recently,

Ton box interactions were studied by NMR (75) and suggested that TonB’s C-

terminal residues form domain-swapped, β-strand interactions upon binding to

Ton boxes.

Global interactions between TonB and FhuA have been the subject of

biophysical characterizations. Stoichiometries and affinities of periplasmic TonB

derivatives bound to FhuA were characterized by AUC and by SPR (77). By

AUC, two periplasmic TonB derivatives possessing either residues 33–239 or C-

terminal residues 155–239 both formed 2:1 TonB–FhuA complexes with FhuA

Page 61: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

37

that were enhanced in the presence of siderophore. SPR analyses further revealed

two TonB populations bound to FhuA: a TonB C-terminal, weaker affinity

population and a TonB N-terminal, higher affinity population. When FhuA bound

siderophore, TonB’s C-terminal affinity decreased and its N-terminal affinity

increased.

Deletion of TonB’s proline-rich region reduced the stoichiometry of the

TonB–FhuA complex to 1:1 (94), but did not alter its affinity for FhuA,

suggesting that the region is essential to form the previously observed, 2:1 TonB–

FhuA complex. Further refined SPR analyses (95) demonstrated that TonB–FhuA

interactions are likely to be sequential; a single TonB monomer is recruited to the

periplasmic surface of FhuA, followed by structural rearrangement that recruits a

second monomer.

Crystal structures of periplasmic TonB fragments bound to FhuA (96) and

to BtuB (97) ultimately demonstrated the determinants of binding between these

three proteins (Figure 1.8). Both structures confirmed findings from earlier

studies suggesting that TonB residue 160 interacted with receptor Ton boxes and

further confirmed that this interaction was through β-strand complementation

between TonB’s C-terminus and Ton boxes of FhuA/BtuB. The structures were

remarkably similar and revealed 1:1 TonB–FhuA and TonB–BtuB

stoichiometries. In both structures, TonB occupies approximately one-half of the

periplasmic cork/β-barrel surfaces. Despite the novelty of confirming the modes

of binding between TonB and periplasmic receptor surfaces, the structures did not

Page 62: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

38

Figure 1.8. Crystal structures of TonB bound to FhuA and BtuB. Crystal structures of TonB–

FhuA and TonB–BtuB are aligned and displayed as ribbon representations. A. Lateral view of the

TonB–FhuA complex (PDB code 2GRX) as viewed from the OM plane. Protein domains are

coloured as follows: FhuA cork (orange), FhuA β-barrel (blue), TonB (cyan). Bound ferricrocin is

displayed as a red surface representation; B. Lumenal view of TonB–FhuA complex as viewed

from periplasm; C. Lateral view of TonB–BtuB complex as viewed from the OM plane (PDB

code 2GSK). Protein domains are coloured as follows: BtuB cork (green), BtuB β-barrel (orange),

TonB (cyan). Bound cobalamin is displayed as a magenta surface representation; D. Lumenal

view of TonB–BtuB complex as viewed from periplasm.

reveal further perturbations that could explain how energy transduction through

TonB elicits structural rearrangement of receptor corks.

Though not resolved in the TonB–FhuA structure, conformations of FhuA

extracellular loops are postulated to change upon binding siderophore or TonB. A

study combining phage display, SPR and fluorescence spectroscopy demonstrated B

A B

Page 63: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

39

conformational changes within FhuA extracellular loops 3, 4 and 5 upon binding

ferricrocin or TonB (98). Since these changes were not observed in apo- and

siderophore-bound FhuA crystal structures, FhuA must exhibit structural

dynamics not accounted for in the crystal structures. These dynamics likely result

in gradual closing of loops over FhuA’s siderophore binding site, similar to the

changes observed for FecA and BtuB upon binding of their respective ligands.

An NMR and infrared spectroscopy study has attempted to characterize

binding between the isolated cork domain of S. typhimurium FepA and E. coli

TonB (56). The FepA cork domain was completely unfolded in solution, yet it

still bound TonB. Interaction did not promote secondary structure formation

within the cork and probably resulted from interactions between FepA’s Ton box

and TonB. However, it is unclear if the lack of TonB–induced cork folding was

due to ortholog incompatibility. From this study, it appears that interactions

between TonB and TBDTs localize primarily to regions accounted for in the

TonB–FhuA and TonB–BtuB crystals structures.

1.7.3 TonB–cell envelope protein interactions

In addition to interacting with TBDTs, TonB has been postulated to

interact with other components of the cell envelope. Formaldehyde cross-linking

studies of fractionated E. coli cell compartments, indicated that TonB formed

complexes with OmpA and Lpp (99). However, the biological significance of

these interactions remains unanswered. Interactions between TonB and Lpp are

particularly interesting considering a recent investigation suggesting that TonB’s

Page 64: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

40

C-terminus possesses a structural motif similar to YcfS, an E. coli proline-rich,

peptidoglycan-binding protein (100). The study demonstrated that, like YcfS,

periplasmic TonB derivatives also bind peptidoglycan. Together, these findings

suggest that C-terminal TonB motifs may enable “surveillance” of the OM inner

leaflet through combinations of interactions with OmpA, Lpp and peptidoglycan,

or perhaps all three.

1.8 Mechanisms of TonB–dependent energy transduction

Currently much is known about structural properties of TonB and the

TBDTs to which it transduces energy. However, there is no consensus

understanding of how TonB transfers energy across the cell envelope. It is

recognized that the TonB–ExbB–ExbD complex harnesses PMF to

conformationally charge TonB, but the molecular details of this process are not

elucidated. Three models of TonB energy transduction are now described: the

shuttle model, the rotation model, and the mechanical pulling model.

1.8.1 The shuttle model

In the shuttle model, TonB is postulated to undergo a process of CM-

localized and PMF-dependent energy excitation by interacting with ExbB and

ExbD (Figure 1.9). TonB remains conformationally charged until a TBDT signals

siderophore-occupancy through extension of its Ton box into the periplasm.

TonB then engages the TBDT and is physically released from the CM, whereupon

it fully engages siderophore-bound TBDT at the OM. Energy release then occurs

Page 65: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

41

through an unknown mechanism and is transduced to the transporter. This causes

rearrangement of the cork and allows siderophore passage into the periplasm.

After transport, TonB is recycled back to the CM, presumably by interacting with

ExbB and ExbD. Evidence for this model originally derives from cell envelope

fractionation studies. While TonB was always detected in the CM fraction, a

significant proportion (~ 40% of total ) could always be detected in the OM (101).

Deletion of ExbB and ExbD prevented recycling of TonB back to the CM and

promoted a population of TonB that was then only detected in the OM fractions.

A second study provided in vivo evidence to support this hypothesis by

engineering a TonB derivative with a single cysteine introduced at TonB’s N-

terminus. During energy transduction, this N-terminal cysteine became labelled

with the thiol-reactive fluorescent dye Oregon green, indicating that TonB’s N-

terminus had escaped the CM and was accessible to labelling (102).

Figure 1.9. Shuttle model of TonB-dependent energy transduction. A. Siderophore binding to an

OM embedded TBDT signals occupancy by extending the receptor’s Ton box into the periplasm.

Within the CM, the TonB–ExbB–ExbD complex harnesses PMF; B. TonB becomes

conformationally charged; C. TonB escapes the CM and engages ligand-bound receptor. By an

unknown mechanism (denoted with a question mark), TonB elicits conformational

changes within the receptor that enables ligand translocation into the periplasm; D. Discharged

TonB disengages the receptor; E. TonB is recycled back to the CM by ExbB/ExbD and the PMF.

Page 66: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

42

The shuttle model has undergone much scrutiny and criticism. In a recent

study, TonB hybrids were generated, which fused green fluorescent protein (GFP)

to either terminus of TonB (100); fusion proteins were expressed at chromosomal

levels in E. coli. One hybrid, possessing an N-terminal, cytoplasm-localized GFP

fusion, exhibited wild-type iron uptake kinetics and was the only hybrid to exhibit

GFP fluorescence. Since GFP fluorescence never decreased during transport, it

was concluded that TonB’s N-terminus never left the CM. This interpretation is

in accordance with reports claiming that periplasmic GFP does not fluoresce

(103). The same study also determined that TonB fractionates equally between

the OM and CM. Taken together, these results shed doubt on the shuttle model by

suggesting that TonB fractionation patterns are artefacts of sucrose-density

fractionation.

1.8.2 The rotation model

The rotation model combines distant sequence homology between ExbB

and MotA and between ExbD and MotB with features of dimeric TonB crystal

structures. Described previously, ExbB and ExbD share sequence homology and

topological features with MotA and MotB of the bacterial stator complex. Both

ExbB/D and MotA/B harness PMF and in the case of MotA/B, proton flow

through these proteins transduces into mechanical energy that rotates the bacterial

flagellum. The rotation model assumes that when bound to a TBDT, TonB causes

rigid body rotation of the transporter’s structural elements that open a pore for

siderophore translocation into the periplasm. Energy transduction would proceed

Page 67: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

43

as TonB–ExbB–ExbD harnesses PMF. The model was originally supported by

observations of a cleft between the tightly intertwined, dimeric interface in the

first TonB crystal structures. The cleft was considered a structural element that

could interact with ligand-bound TBDTs. Presumably, rotation of dimeric TonB

(either from two neighbouring TonB–ExbB–ExbD complexes or from two TonB

monomers associated within a single TonB–ExbB–ExbD complex) would

propagate torsional force that structurally alters the receptor’s cork (Figure 1.10).

This perturbation would then facilitate transport of siderophore into the periplasm.

Figure 1.10. Rotation model of TonB-dependent energy transduction. A. Siderophore binding to

an OM-embedded TBDT signals occupancy by extending Ton box into the periplasm; B. TonB

engages ligand-bound receptor and together with ExbB/ExbD harnesses the PMF. This action

causes rotation of TonB and application of a torsional force that through an unknown mechanism

drives siderophore transport into the periplasm. Although one monomer of TonB is illustrated, the

model can accommodate two monomers of TonB, either within one TonB–ExbB–ExbD complex

or from two neighbouring complexes; C. TonB disengages receptor and returns to its ground-level

energy state.

Page 68: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

44

The rotation model supports mechanisms of TonB-dependent transport

that are independent of TonB’s oligomeric state. Rotation of a TonB monomer

bound to a receptor as observed in the TonB–FhuA and TonB–BtuB crystal

structures could be coupled to the postulated 1:7:2 stoichiometry of the CM-

embedded TonB:ExbB:ExbD complex. Similarly, two neighbouring TonB–

ExbB–ExbD complexes could co-localize such that periplasmic regions of TonB

dimerize and bind to a siderophore-bound TBDT. Rotations of each complex

might still promote structural alterations of the TBDT cork such that siderophore

transport could occur.

1.8.3 The mechanical pulling model

Most recently, a particularly attractive model has emerged, suggesting that

TonB transduces energy to siderophore-bound TBDTs in the form of a

mechanical pulling force. Comparative structural analyses of FhuA, FecA, FepA

and BtuB revealed cork/barrel interface features similar to transient protein–

protein complexes (104). These features include interstitial waters that cushion

each receptor’s cork/barrel interface, which might act as a lubricant that facilitates

removal or unfolding of the cork domain, if sufficient energy were input.

Furthermore, arrangements of each receptor’s cork β-strands are oriented

such that only a modest amount of energy input might be required for unfolding.

These hypotheses derive from single molecule unfolding studies; modest forces

applied perpendicular to the plane of strands in a β-sheet are required to break

inter-strand hydrogen bonds and unfold the sheet (105). In contrast, large forces

Page 69: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

45

applied parallel to the plane of a β-sheet are required to unfold the same β-sheet.

Strands comprising cork domain β-sheets are oriented perpendicular to the OM

plane. Therefore bound TonB could unfold this sheet through application of a

modest pulling force away from the membrane plane (Figure 1.11).

The mechanical pulling hypothesis was simulated in silico by means of

steered-molecular dynamics. TonB’s most N-terminal residue that was resolved

within the TonB–BtuB crystal structure was subjected to a pulling force

perpendicular to the OM plane (106). As the simulation proceeded, TonB

remained bound to BtuB’s Ton box, and BtuB’s cork began to unfold. After

TonB’s N-terminus had retracted nearly 200 Å away from its starting position,

Figure 1.11. Mechanical pulling model of TonB-dependent energy transduction. A. TonB

engages ligand-bound receptor as described for the rotation model; B. TonB exerts a mechanical

pulling force perpendicular to the membrane plane, causing the receptor cork to unfold;

siderophore translocates into the periplasm; C. TonB disengages receptor as in the rotation model

and returns to its ground-level energy state.

Page 70: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

46

a pore large enough to accommodate cobalamin translocation opened. Forces

measured during simulation largely exceed the magnitudes of measured forces

described for other biological mechanisms. However, the simulations

demonstrated the mechanism’s plausibility.

1.8.4 Conformational rearrangement of TBDT cork domains

The mechanisms described above attempt to rationalize how TonB

transduces energy to ligand-bound transporters. The mechanical pulling

mechanism offers insight into how energy transduction might couple to unfolding

of a ligand-bound transporter’s cork domain. However, whether energy

transduction results in complete ejection of the cork from the receptor lumen or if

it causes local unfolding is a controversial topic. Evidence for both possibilities

exists. For example, TonB-dependent transport of the ferric-hydroxamate

siderophore, ferricrocin, was not attenuated when FhuA’s cork was tethered to its

barrel by disulfide bridges (107,108), indicating that FhuA’s cork remains within

the barrel during transport. A local conformational change must open a pore large

enough to allow passage of ferric-siderophore. In a similar study, FepA’s cork

domain was subjected to cysteine mutagenesis and expressed in E. coli (109).

Some, but not all of the cork cysteines were labelled during TonB-dependent

transport. Thus, FepA’s cork may partially unfold during transport. However,

another FepA labelling study indicated that extensive portions of FepA’s cork

become labelled during transport, suggesting that its cork completely ejected from

the barrel (110). These conflicting observations require further investigation.

Page 71: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

47

1.9 Periplasmic siderophore transport

Having crossed the outer membrane, siderophores are delivered to CM-

embedded ATP permeases by periplasmic binding proteins (PBP). In E. coli, the

PBP, FhuD, binds and transports ferric-hydroxamate-type siderophores across the

periplasm (Figure 1.12). Other siderophore-binding proteins include ferric-

enterobactin-binding protein FepB, and ferric-citrate-binding protein FecB. In

addition, the PBP BtuF is required for TonB-dependent transport of cobalamin

across the periplasm (Figure 1.12).

FhuD is a 32 kDa bi-lobal protein comprised of 296 residues. Structures

of FhuD with various bound hydroxamate siderophores revealed similar modes of

binding (111,112); siderophores bind in a shallow solvent-exposed pocket situated

between FhuD’s N- and C-terminal lobes (Figure 1.12). Aromatic and conserved

Tyr and Arg residues line the binding site and form hydrogen bonds with bound

siderophores. Whereas all ferric-hydroxamate-like siderophores occupy the same

binding site, the determinants of binding, such as hydrogen bonding patterns and

hydrophobic interactions between FhuD and ligands slightly differ. These

properties bestow FhuD with a broad ligand-binding specificity and accordingly,

it binds siderophores with modest affinities, in the low micromolar range (113).

The structure of BtuF (Figure 1.12) revealed a similar, FhuD-like fold, yet

exhibited a binding site with contrasting features (114). Both FhuD and BtuF

possess N- and C-terminal lobes bridged by a rigid α-helix. Similarly, BtuF binds

cobalamin in a pocket between these lobes. However, BtuF’s binding site is

comparatively hydrophilic, reflecting the cobalamin-specific binding

Page 72: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

48

determinants. This specificity bestows BtuF with high affinity (Kd ~15 nM) for

cobalamin (115).

1.9.1 Ligand-induced periplasmic binding protein conformational changes

Some periplasmic binding proteins, such as maltose-binding protein

(MBP), exhibit large conformational changes upon binding ligands (116). The

structure of MBP (Figure 1.12) is similar to siderophore binding proteins; it

contains N- and C-terminal lobes with a maltose-binding site in between (117).

Figure 1.12. Periplasmic binding proteins. Various periplasmic binding proteins are displayed as

ribbon representations. A. Ferric-hydroxamate binding protein, FhuD (PDB code 1EFD). Bound

ferrichrome is displayed as a red surface representation; B. cobalamin-binding protein, BtuF (PDB

code 1N4A). Bound cobalamin is displayed as a magenta surface representation; C. apo-maltose

binding protein (PDB code 1N3X); D. holo-maltose binding protein (PDB code 1ANF). Bound

maltose is displayed as a pink surface representation

Page 73: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

49

Comparison of apo-MBP and maltose-bound MBP structures (Figure 1.12) reveal

that MBP’s N- and C-terminal lobes close over bound maltose, excluding it from

solvent (118).

Compared to the structure of ferrichrome-bound FhuD, an unpublished

apo-FhuD structure demonstrated virtually no conformational change (119).

Similarly, an apo-BtuF structure demonstrated only slight reduction of mobility

upon binding cobalamin (120). This conformational change is notably less

pronounced than those associated upon ligand binding of MBP-like PBPs.

Structural elements bridging the lobes of MBP-like PBPs facilitate the large

ligand-induced conformational changes. Three β-strands bridge the lobes of

MBP, whereas rigid α-helices bridge the lobes of siderophore binding proteins. In

MBP, maltose binding exploits the flexibility of this bridge to promote surface

area burial within the ligand binding site. In contrast, rigidity of the α-helical

bridge in siderophore-binding PBPs reduces the degree of surface area burial that

can be afforded when ligands bind.

Despite the conformational rigidity of FhuD and BtuF structures, there is

recent evidence that both proteins exhibit more substantial conformational

changes upon binding ligands. Molecular dynamics simulations of FhuD and

BtuF suggested that there is at least some degree of flexibility in these proteins,

not accounted for in the crystal structures (119,121,122). Furthermore, structures

from two crystal forms of the FhuD-like PBP, FitE, from a clinical E. coli isolate,

also demonstrated a degree of flexibility that was reduced upon ligand binding

A C

C

Page 74: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

50

(123). Further investigation is required to determine if siderophore PBPs exhibit

similar ligand binding mechanisms as MBP-like PBPs.

1.10 Cytoplasmic membrane permeases

Siderophore-bound PBPs ultimately deliver their cargo to CM-embedded

permeases that transport siderophore into the cytoplasm. Permeases belong to the

broad and ubiquitous class of ABC transporters found in all kingdoms of life

(124). ABC transporters comprise two functionally distinct domains: a TM gated

channel and a cytoplasmic nucleotide-binding domain (NBD). Domain

stoichiometries vary; FhuB/C, the ferric-hydroxamate siderophore permease,

comprises a single polypeptide unit (FhuB) that forms the TM channel and two

individual cytoplasmic NBDs (FhuC). In contrast, BtuC/D, the cobalamin

transport permease, comprises a dimer of BtuC chains that form the TM channel

and two cytoplasmic NBDs (BtuD). The BtuC/D organization appears to be the

most common, since homologous ferric-citrate and ferric-enterobactin permeases

(FecC/D and FepC/D respectively) share the dimeric TM domain and NBD

organisation (125).

As prototypical metal-chelate permease, the BtuC/D crystal structure

revealed a representative TM domain organization (126) that is likely shared

amongst homologs. Each BtuC monomer forms a CM-embedded, ten TM helical

bundle. Between the BtuC dimer interface is a gated channel that forms the

cobalamain translocation route. Bound to the cytoplasmic surface of BtuC is a

BtuD NBD dimer. Overall, the BtuC/D structure resembles other ABC

Page 75: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

51

transporter structures, including the putative metal-chelate-type transporter

HI1470/1 from H. influenzae (127), the molybdate transporter ModB/C from

Archaeoglobus fulgidus (128), and the maltose transporter MalFG/K2 from E. coli

(129).

1.10.1 Permease–periplasmic binding protein interactions

Siderophore transport across the CM initiates once a siderophore-bound

PBP docks to its cognate permease. A structure of BtuF bound to BtuC/D

revealed the determinants of binding between a TonB-dependent transport PBP

and its CM-permease (130). Conserved Arg residues on periplasm-exposed

surface loops of BtuC formed electrostatic interactions with conserved Glu

residues on each lobe of BtuF. Negatively charged residues on the lobes of FhuD

are also conserved and a similar binding mechanism may position FhuD on the

surface of FhuB/C. An investigation that mutated homologous residues of

FhuD2, a FhuD ortholog from S. aureus, provided evidence to support this

mechanism (131). Ferrichrome transport was impaired in bacteria harbouring

these mutants, indicating their importance in vivo. The interface between FhuD

and FhuB was further mapped using synthetic FhuB-derived peptides that bound

to FhuD (132); a FhuB peptide corresponding to predicted periplasmic loop 3

bound to FhuD and inhibited ferrichrome transport in vivo, indicating that the

FhuB region interacts with FhuD during transport.

Page 76: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

52

1.10.2 Permeases: transport mechanism

Bacterial ferric-iron acquisition culminates with ferric-siderophore

passage through a permease and into the cytoplasm. Energy for transport across

the CM is provided by cytoplasmic, NBD-mediated ATP hydrolysis. Insight into

permease transport has been provided by structures of various ABC-transporters,

each considered to represent a conformational intermediate within a common

transport mechanism. These structures reveal differently gated transporter

conformations, described as either inward- or outward-facing. In its resting,

nucleotide-free state, the structure of BtuC/D displays an outward-facing

conformation, with its channel opened towards the periplasm (126). In contrast,

the structure of putative metal chelate transporter HI1470/1 revealed an inward-

facing conformation, with its channel open to the cytoplasm (127). The structure

of apo-BtuF bound to BtuC/D revealed an intermediate conformation (130).

Whether a transporter adopts an inward- or outward-facing conformation is

postulated to depend on occupancy of its NBDs (133). Comparisons of different

nucleotide-bound states of other ABC transporters revealed a coupling mechanism

that propagates to the transporters’ TM domains. Upon binding ATP, one NBD

conformationally engages its neighboring NBD. This spatial engagement

propagates through the transporter to yield an outward-facing conformation of its

TM helices. After ATP hydrolysis, ADP is bound and the NBDs disengage. This

conformational change then propagates through the transporter to yield an inward-

facing conformation, and a channel opening directed to the cytoplasm.

Page 77: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

53

Concomitant with nucleotide exchange, ligands delivered by PBPs are considered

to switch their localization from the periplasm to the cytoplasm.

1.10.3 Intracellular fate of iron

Having acquired ferric-siderophores in the cytoplasm, iron is ultimately

removed from the siderophore backbone and processed. Given that iron binds to

siderophore backbones with extremely high affinity, its release requires catalytic

removal. Intracellular esterases, such as enterochelin esterase, hydrolyze

siderophore backbones, causing cytoplasmic release of iron (134). Other iron

release mechanisms involve enzymatic reduction of iron to it ferrous state, which

owing to its weaker affinity, is more easily removed from the siderophore

backbone. FhuF, an E. coli Fe-S cluster-containing enzyme was recently

demonstrated to reduce ferric-hydroxamate siderophores, which led to release of

iron into the aqueous phase (135).

Once released into the cytoplasm, iron is processed into a variety of forms.

One fate involves iron sequestration and storage. Proteins such as bacterioferritin

and mini-ferritin act as iron storage compartments. These proteins oligomerize

into large spherical compartments that bind up to 3000 atoms of iron for ferritin,

and up to 500 atoms for mini-ferritin (136). A second fate involves shuttling iron

into Fe-dependent proteins, such as metabolic enzymes. In addition, the

intracellular iron concentration affects iron-regulated gene transcription.

Transcription of iron-regulated operons, such as those encoding siderophore

transport systems, become de-repressed under iron-deplete conditions.

Page 78: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

54

Conversely, when iron stores are sufficient, iron-regulated gene transcription is

repressed.

1.11 Introduction to techniques used in this thesis

Genetic and biophysical techniques are described in this thesis that

elucidated protein–protein interactions involved in TonB-dependent siderophore

uptake. A brief introduction to each technique is now discussed to facilitate a

greater understanding of the strategies employed to characterize these interactions.

1.11.1 Phage display

Phage display is a molecular genetic technique that predicts and localizes

binding determinants between two macromolecules. Reported applications

include localization of small molecule binding sites on drug targets (137,138),

identification of antibody epitopes (98), and identification/localization of protein–

protein interaction surfaces (139). The latter aspect of phage display plays a

central role in this thesis and is described in greater detail in chapters 2 and 3.

Phage display capitalizes on the vast knowledge of bacteriophage M13

molecular biology. Combinatorial M13 phage libraries are engineered to display

random peptide fusions at the N-termini of various coat proteins. The libraries

described chapters 2 and 3 display up to three copies each of random peptide

fusions at the N-terminus of the pIII minor coat protein. Combinatorial phage

libraries incorporate up to one billion random sequences, each displayed

separately on a single phage particle. Large numbers of sequence displays ensure

Page 79: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

55

that peptide sequence space and peptide conformation space is efficiently sampled

(140).

The process of predicting and localizing protein–protein interactions by

phage display occurs through an iterative process known as phage panning, or

affinity selection (Figure 1.13). The goal of panning is to identify target proteins

with sequences similar to those displayed by phage that specifically interacted

with a selected bait protein. This is accomplished experimentally by incubating

phage libraries with purified bait protein in a well of a microtitre plate. A wash

procedure ensures removal of non-interacting or weakly-interacting phage, while

phage that display bait-interacting peptides are retained. Bait-interacting phage

are then eluted and amplified by infecting an E. coli host strain. Amplified phage

are harvested and represent an enriched population that were selected based upon

their affinity for the bait protein. Amplified phage are generally subjected to

additional rounds of panning to further enrich for a population that displays high

affinity bait-binding peptide sequences.

After subsequent rounds of phage panning, phage DNA is purified and

sequenced; peptide sequences that interacted with the bait protein are identified.

Phage-derived sequences are then compared to target protein sequences by pair-

wise alignments and scored with an alignment matrix. Sequences scoring above a

selected threshold are considered to align significantly with the target protein

sequence. Ideally, many phage-derived sequences align within a region of the

target protein sequence, so as to form a cluster. Clusters of aligned peptides

indicate likely regions of interaction between bait and target proteins.

Page 80: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

56

Figure 1.13. Phage panning. The process of phage panning identifies complementary protein

interaction sequences. A. A combinatorial phage library is incubated with a bait protein that coats

a microtitre well; B. weakly- or non-interacting phage are washed away, leaving only bait-

interacting phage; C. bait-interacting phage are eluted; D. eluted phage are amplified by infection

of host bacteria; E. panning with the enriched phage population is repeated; F. phage DNA is

harvested and sequenced after a few rounds of panning; G. peptides derived from bait-interacting

phage are aligned against target protein(s). Bait-interacting peptides that align within a cluster on

the target sequence define a region of probable interaction between bait and target proteins.

Information from phage panning can refine regions of interaction between

proteins known to interact, or can identify novel protein–protein interactions.

Predictions of novel protein–protein interactions are then demonstrated.

Complementary protein–protein interaction surfaces can be predicted if a previous

target protein is purified and subsequently used as bait for a second phage panning

experiment. These activities enable predictions of interacting regions between

two proteins and guide further experimental strategies to refine complementary

interaction surfaces.

Page 81: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

57

1.11.2 Surface plasmon resonance

The biophysical technique of SPR is a valuable and sensitive tool to

characterize protein–protein interactions in real-time and without need for

labelling. Information such as kinetic association and dissociation rates can be

measured and used to calculate protein–protein binding constants. Furthermore,

information about interaction stoichiometries can be estimated from fits of data to

appropriately selected models.

In SPR, changes in bulk refractive indices that occur when protein

analytes bind to immobilized protein partners are measured and quantified (141).

Immobilization proceeds by covalently tethering a protein (the ligand) to the

surface of a chemically derivatized SPR sensor chip, which is encased within a

microfluidic flow cell. Various chemistries are used to selectively couple and

orient a protein ligand to the sensor chip. These include amine-coupling

chemistries that randomly orient the ligand, or thiol-coupling chemistries that can

directionally orient the ligand. Once immobilized, a protein analyte is then

injected over the ligand surface and responses are recorded.

Interactions between immobilized protein ligand and analyte cause

accumulation of mass on the sensor chip surface. Mass accumulation is optically

detected as a change in the bulk refractive index. Real-time association between

ligand and analyte are recorded in values of resonance units. As mass

accumulates on the sensor chip surface, there is a concomitant increase in the

amount of resonance units recorded.

Page 82: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

58

A second injection of SPR running buffer initiates dissociation of the

ligand–analyte complex. As analytes dissociate from the sensor chip surface, the

accumulated mass decreases and a concomitant decrease in the amount of

resonance units is recorded. Throughout the SPR experiment, changes in bulk

refractive indices that occur in real-time upon association and dissociation of

analyte are recorded and the resulting sensorgrams are fit to an appropriately

selected experimental model.

The choice of model used to fit SPR sensorgrams depends largely on

known information pertaining to the biological system. For example, a model that

describes simple 1:1 binding is selected if two proteins are known to interact in a

1:1 stoichiometry. If other stoichiometries are known, more appropriate models

are selected. When fitting the sensorgrams to a model, rates of association and

dissociation are estimated. These rates are used to calculate the binding constant

for the protein–protein interaction being considered.

1.11.3 Dynamic light scattering

Dynamic light scattering (DLS) is an optical technique characterized by

the unique ways that particles scatter light in solution. In DLS, purified protein is

placed into a sample cell and light is shone upon the sample. Samples might

contain an individual protein or a protein–protein complex. Incident light is

scattered by the protein particles and the scattering intensities are measured over

short time intervals ranging from 100 ns to 30 ms. The magnitude of scattered

light intensity fluctuates with time and arises due to Brownian motion of the

Page 83: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

59

suspended protein particles. The rate that scattered light intensities fluctuate

arises from the translational diffusion coefficient of the protein particles, which is

a function of the protein’s hydrodynamic radius and molecular weight (142).

Since light scattering profiles are related to particle molecular weight, they enable

estimations of protein sizes in solution. This is accomplished by mathematical

deconvolution of scattering profiles into a histogram describing the sizes of

components that could so scatter the light. The ability to measure sizes of

proteins in solution makes DLS a useful tool for investigating protein–protein

complexes. If two proteins interact in solution, the molecular weight

corresponding to the complex should be measurable by DLS.

1.11.4 Analytical ultracentrifugation

Analytical ultracentrifugation (AUC) is a hydrodynamic technique that

complements information provided from DLS measurements. Like DLS, AUC

also enables characterizations of molecular weight and stoichiometry of

macromolecular complexes, and provides information on the shape of a given

protein in solution. This is obtained from measurements of protein sedimentation

profiles. In response to an applied centrifugal force, proteins sediment in ways

that depend on hydrodynamic shape and size (143). Experimentally, this is

followed by measuring absorbance of a protein sample as it sediments. The

protein sample is placed inside a sample cell that is housed inside a specialized

centrifuge rotor. Attached to the rotor is an optics system that monitors the

sample cell’s radial absorbance during the sedimentation experiment; upon

Page 84: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

60

application of centrifugal force, the protein sediments toward the bottom of the

sample cell. Sedimentation is optically monitored by measuring sample

absorbance at 280 nm along the cell’s length, and proceeds until the protein has

sedimented to the bottom of the sample cell.

Resulting sedimentation profiles are then fit to the Lamm equation, which

describes the sedimentation behaviour of a particle in solution. Fits to the Lamm

equation yield information on the sedimentation coefficient of the protein, which

is directly proportional to its molecular weight (144). Additional parameters that

are fit include the protein’s frictional ratio, a measure of its hydrodynamic shape.

Frictional ratios measure deviation of protein hydrodynamic shape relative to that

of a perfect sphere; globular proteins possesses frictional ratio values of around

1.3, whereas elongated proteins possess values around 1.8 or greater.

1.12 Rationale and thesis objectives

TonB is a central element involved in siderophore transport. Upon

starting this project, a wealth of knowledge existed about how TonB interacts with

OM receptors. However, relatively less was known about other periplasmic TonB

interactions. The goals of this project were first, to predict which regions on the

periplasmic surface of FhuA would bind to TonB. This objective is discussed in

Chapter 2. Our use of phage display accurately predicted periplasm-exposed

regions on FhuA that were later confirmed with our X-ray crystal structure of the

TonB–FhuA complex. In addition to the novelty of that structure, it provided

proof of principle that our phage display strategies were revealing true protein–

Page 85: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

61

protein interfaces. Discussed in chapter 3 is the second objective; to predict

additional periplasmic interactions that TonB is capable of making. To our

surprise, the periplasmic binding protein, FhuD, was predicted to bind TonB. We

then demonstrated that TonB and FhuD interacted in vitro. In chapter 4, a third

objective is discussed; to identify essential determinants of binding between TonB

and FhuD. Using biophysical and computational characterizations, we delineated

essential FhuD-binding regions in TonB and identified a plausible mode of

binding that orients FhuD’s siderophore binding site towards the OM, when

bound to TonB. In chapter 5 the final objective of this project is discussed; co-

crystallization of the TonB–FhuD complex. Here, protein preparations and

TonB–FhuD complex formation are discussed. Furthermore, properties of TonB

that are refractory to crystallization were identified. In chapter 6, our

experimental findings are summarized in the context of current models that

describe mechanisms of TonB-dependent transport.

Page 86: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

62

Preface to chapter 2

Chapter 2 describes the use of phage display technology to predict TonB-

binding surfaces on the periplasm-exposed regions of TonB-dependent

transporters. Phage libraries were panned against TonB and sequences derived

from TonB-interacting phage were identified. These were compared to primary

sequences of TonB-dependent transporters and scored through pair-wise

alignments. This strategy predicted regions within the transporters that TonB

would bind to. These regions were subsequently visualized on crystal structures

of E. coli transporters FhuA, FepA, FecA and BtuB. Regions within FhuA that

were predicted to bind TonB were cloned within the N-terminus of maltose-

binding protein. Binding of these FhuA-derived peptides to TonB was confirmed

by ELISA.

Page 87: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

63

Chapter 2

Phage display reveals multiple contact sites between FhuA,

an outer membrane receptor of Escherichia coli, and TonB

David M. Carter, Jean-Nicolas Gagnon, Moussab Damlaj, Suneeta Mandava,

Lee Makowski, Diane J. Rodi, Peter D. Pawelek and James W. Coulton

Journal of Molecular Biology (2006). 357: 236-251

Copyright © 2005, Elsevier Ltd All rights reserved

Page 88: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

64

2.0 Summary

The ferric hydroxamate uptake receptor FhuA from Escherichia coli

transports siderophores across the outer membrane (OM). TonB–ExbB–ExbD

transduces energy from the cytoplasmic membrane to the OM by contacts

between TonB and OM receptors that contain the Ton box, a consensus sequence

near the N-terminus. Although the Ton box is a region of known contact between

OM receptors and TonB, our biophysical studies established that TonB binds to

FhuA through multiple regions of interaction. Panning of phage-displayed

random peptide libraries (Ph.D.-12, Ph.D.-C7C) against TonB identified peptide

sequences that specifically interact with TonB. Analyses of these sequences using

the REceptor LIgand Contacts (RELIC) suite of programs revealed clusters of

multiply-aligned peptides that mapped to FhuA. These clusters localized to a

continuous periplasm-accessible surface: Ton box/switch helix; cork

domain/strand; and periplasmic turn 8. Guided by such matches, synthetic

oligonucleotides corresponding to DNA sequences identical to fhuA were fused to

malE; peptides corresponding to the above regions were displayed at the N-

terminus of E. coli maltose-binding protein (MBP). Purified FhuA peptides fused

to MBP bound specifically to TonB by ELISA. Furthermore, they competed with

ligand-loaded FhuA for binding to TonB. RELIC also identified clusters of

multiply aligned peptides corresponding to the Ton box regions in BtuB, FepA,

and FecA; to periplasmic turn 8 in BtuB and FecA; and to periplasmic turns 1 and

2 in FepA. These experimental outcomes identify specific molecular contacts

Page 89: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

65

made between TonB and OM receptors that extend beyond the well-characterized

Ton box.

2.1 Introduction

Iron is essential in bacteria for processes such as respiration, RNA

synthesis and inactivation of reactive oxygen species (145). Under physiological

conditions, free ferric iron, present at extremely low concentrations (10-18

M),

limits bacterial growth (6). To circumvent this problem, Gram-negative bacteria

have evolved specialized iron transport systems for uptake of siderophores, iron

chelators of less than 1000 Da. These transport systems are composed of a high-

affinity outer membrane (OM) receptor, a periplasmic binding protein and an

ATP-dependent cytoplasmic membrane (CM) transporter. The TonB–ExbB–

ExbD complex provides energy required by the OM receptors for unidirectional

transport of siderophores (146). Anchored in the CM, this complex harnesses

energy of the proton motive force. Given that TonB physically associates with

OM receptors in vivo, it is likely that these interactions result in energy

transduction necessary for ligand transport (89,90).

One paradigm of a TonB-dependent OM receptor is FhuA, receptor for the

siderophores ferrichrome and ferricrocin. In addition, FhuA serves as receptor for

phages T1, T5, 80 and UC-1, the siderophore-antibiotic conjugate albomycin,

rifamycin CGP 4832, the bacterial toxin colicin M, and the antimicrobial peptide

MccJ21 (147-149). With the exception of phage T5, uptake of these ligands

requires energy from the TonB–ExbB–ExbD complex for transport. X-ray

Page 90: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

66

crystallographic studies of the TonB-dependent E. coli OM receptors FhuA

(42,150), FepA (44), FecA (45), BtuB (46), and the OM receptors FpvA (52) and

FptA (51) from Pseudomonas aeruginosa revealed common structural features.

All six receptors are composed of a C-terminal 22-stranded -barrel domain and

an N-terminal domain termed the cork that occupies the interior of the barrel. In

FhuA, the cork tightly interacts with the interior of the barrel via a network of

hydrogen bonds and salt bridges (42). Recent analyses (104) of available

structures of TonB-dependent OM receptors indicates that the cork-barrel

interface is highly hydrated. Presence of a large number of interfacial water

molecules may reduce the energy required to effect subtle conformational changes

or even significant movements of the cork domain, either of which could facilitate

siderophore transport (151).

The first crystal structure of the C-terminal domain of TonB (residues

155–239) (72) provided unexpected evidence that TonB forms a dimer. The

authors speculated that a region of the dimer interface proximal to residue Asn-

200 forms a binding cleft for OM receptors. Based on their structural data, they

proposed a mechanism whereby the proton motive force provides propeller-like

torsional motion of dimeric TonB, thereby inducing conformational change in

OM receptors. An alternate model postulated that TonB shuttles between the CM

and the OM (102,152). In the shuttle model, TonB may dissociate from the CM

after having been energized by ExbB–ExbD, delivering stored potential energy to

OM receptors and allowing transport of ligands into the periplasm. Two

additional structures from progressively longer C-terminal TonB constructs have

Page 91: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

67

since been reported. An X-ray crystallographic structure of TonB 148–239

indicated that this protein was dimeric, although much more loosely packed than

the dimer formed by TonB 155–239 (74). Most recently, an NMR structure of

TonB 103–239 demonstrated a monomer in solution (75).

The oligomeric state of TonB when in contact with OM receptors has

recently been investigated by numerous methods. Our analytical

ultracentrifugation studies (77) of TonB-FhuA interactions identified a 2:1

complex. These findings were corroborated by in vivo studies using a bacterial

two-hybrid system (78). However, Koedding et al. (73) proposed that TonB

dimerization is not necessary since in vivo, TonB constructs that could block

TonB-dependent processes were shown by analytical ultracentrifugation to be

monomeric in solution. Even though Khursigara et al. and Koedding et al. draw

different conclusions from their observations, the two studies may be reconciled

since both groups determined that the C-terminal portion of TonB forms a dimer

in solution; longer TonB constructs (composed of at least residues 145–239) are

monomeric in solution, consistent with the reported NMR structure. Most

recently, Ghosh and Postle (79) demonstrated TonB dimerization at the CM in a

manner requiring both the TonB signal anchor sequence and energy input from

the ExbB–ExbD complex. They found monomeric TonB at the OM, indicating

that dynamics of TonB oligimerization during ligand transport remain only

partially understood.

All TonB-dependent OM receptors share a conserved five to six amino

acid sequence near their amino termini, a region termed the Ton box that is

Page 92: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

68

proposed to interact with TonB during energy transduction. Mutations in this

region abolished TonB-dependent activities of these receptors; TonB-independent

receptor-mediated activities remained unaffected (153,154). Gudmundsdottir et

al. (55) demonstrated that rather than strict amino acid sequence, conformation of

the Ton box is important for its specific interaction with TonB; mutations that

were most deleterious to normal functions of BtuB were those that introduced

glycine or proline into the Ton box. Other amino acid substitutions in the same

region had little effect on TonB-dependent activities. Another study showed that

a mutation (Ile14Pro) in the Ton box of FepA resulted in total loss of TonB-

dependent activities (91). In addition, insertion of cysteine residues in the Ton

box of BtuB (92,93) or FecA (155) and insertion of cysteine residues around

amino acid 160 of TonB resulted in disulfide bridge formation between these two

protein partners. Finally, binding of vitamin B12 to BtuB triggers a

conformational change in the Ton box, a conclusion supported by site-directed

spin-labelling (156), biotin maleimide labelling (157) and X-ray crystallography

(46).

Some reports originally proposed that regions other than the Ton box

participate in interactions of OM receptors with TonB because deletions of the

cork domain including the Ton box in FhuA (158) and FepA (159) did not

completely abolish TonB-dependent transport processes. By synthesizing

periplasm-directed TonB fragments, Howard et al. (160) demonstrated reduced

TonB activity in FhuA and corkless FhuA variants alike. Killmann et al. (161)

also observed reduced TonB activity in FhuA and corkless FhuA via interference

Page 93: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

69

from synthetic nonapeptides corresponding to regions near glutamine160 of

TonB. To resolve contradictions, Vakharia and Postle (162) as well as Braun et

al. (163) clearly demonstrated that the phenotype observed for the FepA and

FhuA cork deletions was due to interprotein complementation. However, these

results do not exclude the possibility that TonB interacts with regions other than

the cork of OM receptors since FepA mutants that lack a cork domain co-

precipitated with TonB (162) suggesting weak interactions in addition to the Ton

box residues.

TonB has been shown to interact differentially with OM receptors

(78,164). Mutation in the C-terminal portion of TonB resulted in diverse

phenotypes with respect to TonB-specific processes. In both studies, results were

interpreted by proposing distinct interactions of TonB with each OM receptor.

Our recent studies (77,94,95) investigating TonB-FhuA interactions using

surface plasmon resonance (SPR) showed that TonB possesses two affinities for

FhuA. A low-affinity binding site is located in the C-terminal segment of TonB

between residues 155–239; a higher affinity binding site becomes accessible when

residues 33–154 are present. Consistent with the hypothesis of multiple physical

interactions for TonB with OM receptors, our schematic model for mechanism

incorporated two binding sites for TonB.

Using phage display of peptides as an experimental strategy, we now

report identification of novel TonB-binding motifs in addition to the well

characterized Ton box. These motifs map to a continuous surface along the

periplasmic face of FhuA. FhuA sequences corresponding to potential TonB-

Page 94: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

70

interacting regions identified by phage display were shown to bind specifically to

TonB in vitro. To our knowledge, this is the first application of phage display in

identifying protein–protein interactions within the bacterial cell envelope. These

findings provide substantive evidence for interactions between TonB and OM

receptors that extend beyond the Ton box.

2.2 Materials and methods

2.2.1 Bacterial strains and media

The phage libraries Ph.D.-12 and Ph.D.-C7C were purchased from New

England Biolabs (NEB). E. coli K-12 ER2738 (NEB) was used for amplification

and titration of phage M13 pools; E. coli DH5α was used for cloning of peptide-

coding DNA sequences into pMal-pIII, a vector (165) obtained from C.J. Noren.

[Peptide-MBP] fusions were expressed in E. coli NM522 (NEB). Cultures were

grown in Luria Bertani (LB) broth supplemented with 0.2% [w/v] glucose for

expression of [peptide-MBP] fusions.

2.2.2 Chemicals and reagents

5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) and

isopropyl-ß-D-thiogalactopyranoside (IPTG) were purchased from Biovectra

(Charlottetown, PE, Canada). Protein-grade Tween-20 was from Calbiochem.

All antibiotics used in this study were obtained from Sigma-Aldrich.

Oligonucleotides were synthesized at the Sheldon Biotechnology Centre, McGill

Page 95: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

71

University. Ni-NTA resin used for purifications of H6.'TonB and FhuA was from

Qiagen and amylose resin for purification of [peptide-MBP] fusions was from

NEB.

2.2.3 Protein purification

The TonB construct referred to as H6.'TonB was described by Moeck and

Letellier (166); the first 32 amino acids corresponding to the transmembrane

anchor were removed and replaced by a 20 residue linker including a

hexahistidine tag. The genetically engineered FhuA protein (167) contains a

hexahistidine tag at position 405 within a surface-exposed loop. Both proteins

were purified as previously reported (77).

2.2.4 Phage M13 titre

Plaque assays were performed to determine phage titres. LB plus

tetracycline (20 µg ml-1

) was inoculated with E. coli ER2738 and grown at 37°C

until mid-logarithmic phase. Agarose top (0.7% agarose [w/v] in LB) was melted

and dispensed in 3 ml aliquots and kept at 45°C. Ten-fold serial dilutions of

phage were prepared. E. coli ER2738 (100 µl) was mixed with 100 µl of phage

dilutions, followed by 10 min incubation. Infected cells were transferred to

agarose top and poured onto pre-warmed LB/X-Gal/IPTG plates. After overnight

incubation at 37°C, blue plaques were counted to determine phage titres.

Page 96: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

72

2.2.5 Panning procedures

H6.'TonB was diluted to 100 µg ml-1

in Tris buffered saline (TBS; 50 mM

Tris-HCl, 150 mM NaCl, pH 7.5) and 150 µl was adsorbed to six wells of a 96-

well polystyrene microtitre plate (Nunc Maxisorp). The plate was incubated at

4°C overnight. Wells were blocked by adding 400 µl of blocking buffer (TBS + 5

mg ml-1

BSA + 0.02% [w/v] NaN3) and incubating the plate 2 h at 37°C. They

were washed six times with TBST (TBS + 0.1% Tween-20 [v/v]). Ten µl of

phage library Ph.D.-12 (1.5 × 1011

pfu) or Ph.D.-C7C (1.5 × 1011

pfu) was diluted

to 1 ml in TBST and 120 µl was dispensed in each well. The plate was incubated

for 1 h at room temperature with gentle agitation. Unbound phages were removed

from the wells by washing ten times with TBST. Bound phages were eluted by

100 µl of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg ml-1

BSA). After 10

min incubation at room temperature, the eluates were pooled to one tube

containing 90 µl of 1 M Tris-HCl (pH 9.1) for neutralization. Titres of eluted

phages were determined as described above. The phage pool was amplified by

following the manufacturer’s instructions provided with the Ph.D. libraries

(NEB). This panning procedure was repeated for a total of three rounds for the

Ph.D.-12 library and four rounds for the Ph.D.-C7C library. For each subsequent

round the input number of phages was 1.5 × 1011

pfu for the Ph.D.-12 library and

2 × 1011

pfu for the Ph.D.-C7C library. Stringency of selection was increased by

using 0.3% [v/v] Tween-20 in TBS for the second round and 0.5% [v/v] Tween-

20 in TBS for subsequent rounds.

Page 97: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

73

2.2.6 Isolation of phage M13 clones, DNA isolation and sequencing

An overnight culture of E. coli ER2738, diluted 1/100 in LB plus

tetracycline, was dispensed in 1 ml aliquots. Phage plaques from titration of the

final round of selection were stabbed using a pipette tip and inoculated into 1 ml

E. coli ER2738. The cultures were incubated 4.5 h at 37°C, then transferred to

microcentrifuge tubes and centrifuged at 10,000 × g for 2 min to pellet bacterial

cells. Supernatants were decanted. NaN3 (0.02%) was added to each stock and

titres were determined by plaque assays. Single-stranded DNA from each clone

was isolated using Spin M13 Kit (Qiagen). Quality of isolated DNA was assessed

by agarose gel electrophoresis. DNA samples were sequenced at the Sheldon

Biotechnology Centre, McGill University and at the Genome Québec Innovation

Centre.

2.2.7 Global analysis of affinity-selected peptides

After isolating and sequencing affinity-selected phage from both the

Ph.D.-12 and Ph.D.-C7C libraries, the deduced peptides were statistically

analyzed using the REceptor LIgand Contacts (RELIC) bioinformatics server

(http://relic.bio.anl.gov) (168). The program POPDIV was used to calculate the

diversity of affinity-selected peptides from both libraries. The program INFO was

used to calculate the information contents associated with affinity-selected

peptides. For all analyses, randomly selected peptides were provided by RELIC.

Page 98: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

74

2.2.8 Cloning of peptide-coding DNA sequences into pMal-pIII vector

Sequences identical to fhuA were cloned to create N-terminal fusions to

MBP. Oligonucleotides corresponding to the coding and non-coding strands of

the Ton box (6EDTITVTAA

14), Fhu switch (

21AWGPAAT

27), Fhu cork1

(143

SSPGGLL149

), Fhu cork2 (146

GGLLNMVSK154

), Fhu cork3

(153

SKRPTTEPL161

) and Fhu turn (583

AKAALSA589

) were synthesized at Sheldon

Biotechnology Centre. Design of the oligonucleotides included ends compatible

for ligation into EagI/Acc65I-digested pMal-pIII vector plus a linker region

encoding the peptide GGGS that separates the FhuA sequence from the N-

terminus of MBP. Oligonucleotides were denatured at 95oC for 10 min and

annealed by cooling overnight to room temperature. Ligation required incubation

of annealed products with the expression vector pMal-pIII (Zwick et al. (165))

previously digested with EagI and Acc65I. Ligated products were transformed

into E. coli DH5α and the resulting plasmids sequenced (Sheldon Biotechnology

Centre) to verify faithful incorporation of fhuA inserts.

2.2.9 Peptide-MBP expression

E. coli NM522 harbouring the pMal-pIII derivatives for expression of the

peptide sequences were grown in LB broth supplemented with 0.2% glucose to

mid-logarithmic phase. Expression of [peptide-MBP] fusions was induced by

adding IPTG to a final concentration of 0.2 mM. The culture was grown for an

additional 3 h, after which cells were harvested by centrifugation. Periplasmic

Page 99: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

75

extract was obtained by cold osmotic shock. Cells were suspended in 500 ml of

30 mM Tris-HCl, 1 mM EDTA, 20% sucrose, pH 8.0 and stirred for 20 min, room

temperature. Cells were pelleted by centrifugation, resuspended in 500 ml of ice-

cold 5 mM MgSO4, and stirred for 30 min at 4°C. Cells were pelleted by

centrifugation. The supernatant (cold osmotic shock fluid) was decanted and

filtered through a 0.45 μm filter to remove residual contaminant cells; 10 ml of

1 M Tris-HCl, pH 7.4 were added to the solution. The cold osmotic shock fluid

was concentrated to 40 ml using a YM10 ultrafiltration membrane (Millipore)

before loading onto an amylose column. For elution from the amylose column,

buffers were supplemented with 10 mM maltose. After [peptide-MBP] fusions

were purified and confirmed by SDS-PAGE, they were subjected to N-terminal

protein sequencing (Sheldon Biotechnology Centre); there were no ambiguities,

mismatches, or cleavages of peptide sequences.

2.2.10 Enzyme linked immunosorbent assay (ELISA)

H6.'TonB (20 pmol) was coated into each well of a Qiagen Ni-NTA

HisSorb 96-well microtitre plate and incubated overnight at 4oC. Use of these

plates increased sensitivity of the assay compared to polystyrene (Nunc Maxisorp)

plates. Binding of TonB was accomplished by attachment of its N-terminal

hexahistidine tag to the No-NTA-coated plate. Plates were blocked by addition of

2.5% casein [w/v] in Tris-buffered saline (TBS) and incubated for 3 h at room

temperature, followed by three washes with TBS. MBP fusion proteins (200

pmol) were added to each well and incubated at room temperature for 1 h with

Page 100: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

76

shaking. MBP was probed by addition of anti-MBP antibodies (NEB) followed

by 1 h incubation. Anti-MBP was detected by addition of a secondary goat anti-

mouse IgG coupled to alkaline phosphatase (Cedarlane). Non-specifically bound

proteins were removed after each step by three washes with TBS containing

0.05% Tween-20 (Calbiochem). Plates were developed (37oC) by addition of p-

nitrophenyl phosphate (PNPP, MP Biomedicals). Endpoint absorbance

measurements (405 nm) were taken at 3 h. Raw absorbance data were corrected

by subtraction of a PNPP-containing blank. Competition between ferricrocin-

bound FhuA and MBP constructs for TonB was used as a measure of binding

specificity. For these measurements, equimolar amounts of FhuA and MBP

fusion proteins (200 pmol each) were mixed and added to H6.'TonB-coated wells.

Development and detection of ELISA signals were performed as above.

2.3 Results

2.3.1 Isolation of affinity-selected peptides by phage panning

A TonB construct containing residues 33–239 of E. coli TonB plus an N-

terminal hexahistidine tag (hereafter referred to as H6.'TonB) was assessed by

phage panning for interactions with randomly displayed peptides. Two different

peptide libraries display fusions at the N-terminus of the M13 bacteriophage

minor coat protein, pIII. The Ph.D.-12 library contains a linear dodecamer

separated by a linker; the Ph.D.-C7C library contains a heptapeptide sequence

constrained by a disulfide to form a loop. To enrich for affinity-selected peptides

Page 101: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

77

against TonB, biopanning experiments were conducted. Each library was

incubated with H6.'TonB immobilized in wells of a microtitre plate to allow

retention of TonB-binding phages during washes. Affinity-selected phages were

then eluted. To select for unconstrained peptides that could bind to TonB, we

used the Ph.D.-12 library. The selection procedure was repeated for three rounds;

phage titres were determined after each round of selection. Titres of Ph.D.-12-

eluted phage increased approximately 70-fold over three successive rounds of

panning: 2.0 × 105 pfu ml

-1 (round 1) to 1.37 × 10

7 pfu ml

-1 (round 3). Panning

was not extended after the third round due to accumulation of redundant peptide

sequences. To select for constrained peptides that could bind to TonB, we

repeated our panning procedure with the Ph.D.-C7C library. The selection

procedure was repeated for four rounds. Following each round, the phage titre

ranged from 1.3 to 1.7 × 107 pfu ml

-1 and increased to 1.5 × 10

8 pfu ml

-1 from the

fourth round.

After panning the two libraries, 227 Ph.D.-12 phage clones and 271 Ph.D.-

C7C phage clones were isolated; their single-stranded DNA was purified and

sequenced. The peptide displayed by each phage was determined. From the 498

phage clones, we obtained 105 unique Ph.D.-12-derived sequences and 135

unique Ph.D.-C7C-derived sequences.

2.3.2 Global analysis of affinity-selected peptides

The RELIC suite of programs evaluates statistical properties of affinity-

selected peptides (168). RELIC confirmed that our ensembles of phage-derived

Page 102: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

78

peptide sequences resulted from affinity to immobilized H6.'TonB. One indicator

of affinity selection is reduction in the diversity of sequences obtained from

iterative rounds of affinity selection (140,168,169). In this context, diversity is a

unit-less measure of the relative proportions of the 20 possible amino acids

observed within peptide sequences of a given population. Numerical methods

based on limited sequence data were developed to assess the diversity of a phage

population. Selection of peptides due to affinity for the immobilized target should

yield a population of peptides whose diversity is less than an equivalent number

of randomly-selected peptides from the parent library. This was evident for the

affinity-selected peptides obtained from biopanning against immobilized

H6.'TonB. RELIC/POPDIV, a program from the RELIC suite, determined that

the 105 unique affinity-selected peptides from the Ph.D.-12 parent library yielded

a diversity value of 0.004 ± 0.002; the 135 unique peptides from the Ph.D.-C7C

parent library yielded a diversity value of 0.066 ± 0.022. In contrast, ensembles

of 100 randomly selected peptides from each of the Ph.D.-12 and Ph.D.-C7C

libraries were shown to have diversities of 0.040 ± 0.016 and 0.079 ± 0.026,

respectively (140). The Ph.D.-12 affinity-selected peptides clearly contain a less

diverse population compared to randomly selected peptides from the same library,

while diversity of the Ph.D.-C7C affinity-selected peptides is not significantly

different from randomly selected peptides of the same library. The values

obtained for the H6.'TonB-affinity-selected peptides are comparable to those

obtained for Taxol-affinity-selected peptides (Ph.D.-12: 0.011 ± 0.007; Ph.D.-

Page 103: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

79

C7C: 0.053 ± 0.021), and gamma-ATP-affinity-selected peptides (Ph.D.-12: 0.011

± 0.003; Ph.D.-C7C: 0.065 ± 0.011) (137,168).

Another indicator of affinity selection is a positive shift in the information

content of an ensemble of affinity-selected peptides compared to randomly

selected peptides. Information is defined as –ln(PN) where PN refers to the

probability of observing any given peptide within a parent library by chance alone

(140). Commonly occurring peptides within the parent library have a high

probability of being observed and consequently possess relatively low information

content. Conversely, rare peptides are less likely observed and are associated

with relatively higher information content. Affinity selection yields an ensemble

of peptides in which the distribution of information content is positively shifted

compared to randomly selected peptides. We observed such shifts for our

ensembles of affinity-selected peptides retrieved from biopanning against

immobilized H6.'TonB. Distributions of information content were calculated by

RELIC/INFO for affinity-selected peptides. For both libraries, significant

positive shifts in information content were evident compared to randomly selected

peptides (data not shown). Our affinity-selected peptide sequences therefore

reflect an enrichment of phage-borne peptides in which selection was primarily

due to affinity to immobilized H6.'TonB. Based upon these analyses, it is

unlikely that biases due to non-specific interaction between the phage and

H6.'TonB or factors based upon advantageous phage growth contributed

significantly to the selected peptides.

Page 104: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

80

2.3.3 Identification of TonB-binding sites on the periplasmic surface of FhuA

To identify potential TonB-binding sites on the periplasm-exposed surface

of FhuA, we analyzed the ensembles of TonB-binding peptides isolated from the

Ph.D.-12 and Ph.D.-C7C libraries for similarities to the FhuA sequence. Given a

set of affinity-selected peptides obtained by phage display, the RELIC/MATCH

program identifies potential binding regions within the primary sequence of a

target protein that are most similar to affinity-selected peptides (168). Using a

variant of the BLOSUM62 substitution matrix, affinity-selected peptide sequences

are aligned to each residue within the target protein sequence and evaluated for

pairwise similarity within a scoring window. A peptide in a given position that

scores above a cutoff value (13 in the case of a 5-residue scoring window) is

considered to align to the protein at that position (168) and clusters of aligned

peptides may correlate with sequences involved in binding to the molecular target

(137).

The ensembles of 105 Ph.D.-12 peptides and 135 Ph.D.-C7C peptides

isolated from TonB-binding phages were evaluated by RELIC/MATCH for their

ability to align to the primary sequence of FhuA. Analyses focused on surfaces of

FhuA potentially exposed to the periplasmic space: strand regions of the β-barrel

that are located below the aromatic amino acid girdle, periplasm-exposed β-barrel

turns, and surfaces of the cork domain and barrel lumen accessible to the

periplasm. Given these constraints, we observed three regions in which

RELIC/MATCH identified multiple peptides that align to periplasm-exposed

surfaces of FhuA: the Ton box/switch helix (region I); the cork/1 strand

Page 105: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

81

(region II); and periplasmic turn 8 (region III) (Figure 2.1). The Ph.D.-12 and

Ph.D.-C7C peptides that aligned to the FhuA Ton box overlapped residues from

the Ton box itself, and overlapped residues immediately C-terminal to the Ton

box that coincide with entry into the FhuA switch helix. Peptides aligned to the

FhuA cork domain could be similarly partitioned into two sub sites: the C-

terminal half of cork domain β strand D, and a contiguous region that comprises a

shallow loop entering into β1 of the barrel domain. In contrast, peptides aligned

to turn 8 localized to a single sub site comprised of residues within the

periplasmic turn (Figure 2.1).

Nine peptides from the Ph.D.-12 library were found to align to FhuA with

RELIC/MATCH scores of 13 or greater within a 5-residue scoring window (Table

2.1). Three peptides aligned with scores greater than or equal to 14 within a 7-

residue scoring window. The three highest-scoring Ph.D.-12 peptides aligned to

the FhuA cork domain. The lowest-scoring Ph.D.-12 peptides aligned to turn 8,

indicating that unconstrained peptides from the Ph.D.-12 library more effectively

mimic extended regions in the FhuA cork and Ton box than a periplasmic turn of

the barrel domain. Of the six Ph.D.-C7C peptides identified (Table 2.2) to align

within these same regions, three aligned with a RELIC/MATCH score of 13 or

greater within a 5-residue scoring window. Three additional peptides aligned

with a score of 13 within a 6-residue scoring window. The highest-scoring Ph.D.-

C7C peptide, ILAALSA, matched turn 8 (583

AKAALSA589

) with a score of 20,

consistent with the constrained nature of the Ph.D.-C7C peptides.

Page 106: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

82

Similarity scores for each residue within FhuA scanned against the

ensemble of Ph.D.-12 affinity-selected peptides were determined using the

program RELIC/HETEROalign and displayed along the surface of FhuA (Figure

2.2A). Two regions potentially contributing to TonB-binding sites were

identified. A region shaded in blue-green corresponds to the Ton box/switch

region of FhuA (Figure 2.2D, region I). The region of highest similarity, shaded

in green, is similarly exposed to the periplasm and corresponds to the broad loop

(residues 153–160; Figure 2.2D, region II) connecting the C-terminus of the cork

domain to the β1 strand of the barrel domain. RELIC/HETEROalign similarity

scores corresponding to the Ph.D.-C7C ensemble of peptides were also

superimposed on the periplasmic surface of FhuA (Figure 2.2B). In this case, the

highest-scoring region shaded in red corresponds to periplasmic turn 8 (Figure

2.2D, region III). An additional region of high similarity (green) connected to

region II and consisting of residues from FhuA periplasmic turn 1 was also

observed. When the RELIC/HETEROalign scores from both ensembles were

averaged and displayed on the periplasmic surface of FhuA (Figure 2.2C), we

observed an almost continuous TonB-binding region: originating at turn 8 (Figure

2.2D, region III), spanning the Ton box/switch region (Figure 2.2D, region I), and

extending to the junction of the cork and barrel domains (Figure 2.2D, region II).

Page 107: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

83

Figure 2.1. Alignments of affinity-selected peptides to FhuA as identified by RELIC/MATCH.

A. Region I: Ton box/switch helix; B. Region II: cork/1 strand; C. Region III: periplasmic turn 8.

Peptides from the Ph.D.-12 library are aligned below the FhuA sequence; peptides from the Ph.D.-

C7C library are aligned above the FhuA sequence. See Tables 2.1 and 2.2 for peptide match scores

and window sizes. Alignment positions are highlighted according to their pairwise alignment

score: +4, black background and white character; +1, grey background and dark character.

Page 108: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

84

Table 2.1. RELIC/MATCH identification of TonB-affinity-selected Ph.D.-12 peptides

corresponding to FhuA sequences.

Peptidea

Peptide match

scoreb

Scoring window

(residues) Region

Alignment

positionc

NLLPTYTPLKLM 18 7 cork 156 HSNLPTKRPTSL 17 5 cork 153 CPNGLLGPCPSL 16 5 cork 145 KVWSLEPPGPAA 15 5 Ton box 22 TQPLGLLPSRHL 15 5 cork 145 IPHHHTLNMESH 15 5 cork 149 KPPSTWPQNALH 15 7 cork 154 LETRTTPPAKSQ 14 7 Ton box 10 KLVDESSTSPLS 13 5 cork 157 AGMATSRSTSPL 13 5 cork 157 KHVPVHSALSVN 13 5 turn 8 585 LEPQINSVGMVR 13 5 turn 8 590

aResidues contained in Scoring Window shown in bold.

bSum of pairwise comparisons of aligned residues within the Scoring Window.

cPosition of first residue of the Scoring Window numbering from the N-terminus of the

mature protein.

Table 2.2. RELIC/MATCH Identification of TonB-affinity-selected Ph.D.-C7C peptides

corresponding to FhuA sequences.

Peptidea

Peptide match

scoreb

Scoring window

(residues) Region

Alignment

positionc

ILAALSA 20 5 turn 8 585 LPTGGLL 15 5 cork 145 LTRSPAA 13 6 Ton box/switch helix 11 KSAFLPW 13 6 Ton box/switch helix 19 TKPLTQQ 13 6 cork 158 LAALKST 13 5 turn 8 585

a Residues contained in Scoring Window shown in bold.

b Sum of pairwise comparisons of aligned residues within the Scoring Window.

c Position of first residue of the Scoring Window numbering from the N-terminus of the mature

protein.

Page 109: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

85

2.3.4 Identification of potential TonB-binding sites in structurally conserved OM

receptors

In addition to identifying three regions along the periplasm-exposed

surfaces of the FhuA cork and barrel domains, RELIC/MATCH analyses

identified TonB affinity-selected peptides that multiply align to corresponding

regions in BtuB, FecA, and FepA. From the Ph.D.-12 library, 16 peptides were

identified by RELIC/MATCH to align. Of the seven highest-scoring Ph.D.-12

peptides (score ≥ 15), five aligned to the Ton box regions in BtuB, FecA, and

FepA; two peptides aligned to periplasmic turns (Table 2.3). From the Ph.D.-C7C

library, 17 peptides were identified and aligned to BtuB, FecA, and FepA (Table

2.4). The seven highest-scoring Ph.D.-C7C peptides (score ≥ 15) aligned to

periplasmic turns in the barrel domains of all three proteins, consistent with the

constrained nature of the peptides.

RELIC/MATCH aligned affinity-selected peptides to periplasm-exposed

regions of BtuB that clustered in a region immediately C-terminal to the BtuB

Ton box, as well as at periplasmic turn 8 (Figure 2.3A). The BtuB Ton box

cluster is comprised predominantly of peptides from the Ph.D.-C7C library, as is

the BtuB turn 8 cluster. RELIC/HETEROalign similarity scores mapped to the

periplasm-exposed surface of BtuB clearly identify the BtuB Ton box (Figure

2.4A, region I) and turn 8 (Figure 2.4A, region II) as the highest-scoring regions.

Page 110: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

86

Figure 2.2. RELIC/HETEROalign similarity scores mapped to the periplasm-exposed surface of

FhuA (PDB code 2FCP). Molecular surfaces rendered by PyMOL (170). Normalized

RELIC/HETEROalign similarity scores are represented on surfaces as colour ramps (blue=30 <

green =75 < red=120). A. Ph.D.-12 similarity scores; B. Ph.D.-C7C similarity scores; C. average

of Ph.D.-12 and Ph.D.-C7C similarity scores; D. superposition of the FhuA ribbon representation

and the molecular surface mapped in panels A to C. Regions corresponding to RELIC/MATCH

alignment clusters shown in Figure 2.1 are indicated by Roman numerals: (I) Ton box/switch

helix; (II) cork/1 strand; and (III) periplasmic turn 8.

Two clusters of affinity-selected peptides map to similar regions on the

OM receptor FecA. The FecA Ton box cluster of four Ph.D.-12 peptides and two

Ph.D.-C7C peptides occurs in a region immediately N-terminal to the FecA Ton

box (Figure 2.3B). The highest-scoring Ph.D.-12 peptide, containing the

sequence WSLEP within a 5-residue scoring window, was found to align to

Page 111: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

87

Table 2.3. RELIC/MATCH Identification of TonB-affinity-selected Ph.D.-12 peptides

corresponding to BtuB, FecA, and FepA sequences.

Peptidea

Peptide match

scoreb

Scoring window

(residues) Protein Region

Alignment

positionc

TMGFTAPRFPHY 15 5 BtuB Ton box 11 KLVDESSTSPLS 13 5 BtuB turn 8 472 KVWSLEPPGPAA 17 5 FecA Ton box 69 AALGTYSTHTPT 15 5 FecA turn 8 596 SSMKVWSLPPAP 15 5 FecA Ton box 71 NLLPTYTPLKLM 14 6 FecA turn 8 594 NNSQKPAPVSPF 13 5 FecA Ton box 72 GNSVNKTWTHDY 13 5 FecA Ton box 66 KHVPVHSALSVN 13 5 FecA Ton box 80 SLKNYPVSWKNT 15 7 FepA Ton box 7 NLLPTYTPLKLM 15 5 FepA Ton box 3 TQPLGLLPSRHL 15 5 FepA turn 1 180 HSNLPTKRPTSL 14 7 FepA Ton box 3 QSPVNHHYHYHI 14 5 FepA Ton box 6 KLVDESSTSPLS 14 6 FepA turn 1 183 SHSNTTQTRPSD 13 5 FepA Ton box 9 a Residues contained in Scoring Window shown in bold.

b Sum of pairwise comparisons of aligned residues within Scoring Window.

c Position of first residue of Scoring Window relative to the N-terminus of the mature protein.

Table 2.4. RELIC/MATCH Identification of TonB-affinity-selected Ph.D.-C7C peptides

corresponding to BtuB, FecA, and FepA sequences.

Peptidea

Peptide match

scoreb

Scoring window

(residues) Protein Region

Alignment

positionc

TKPLTQQ 15 5 BtuB turn 8 472 TGPLPNR 15 5 BtuB turn 8 472 SPRTTPF 13 5 BtuB Ton box 17 MLEKPRL 13 5 BtuB Ton box 15 NQPRGPQ 12 4 BtuB Ton box 16 LTQTPTR 12 4 BtuB turn 8 475 LTQTPTR 15 5 FecA turn 8 596 HATLPPT 15 5 FecA turn 8 596 SWDPAPL 13 4 FecA Ton box 72 TLSPKLH 12 4 FecA Ton box 70 TLSPKLH 12 4 FecA turn 8 596 TGPLPNR 15 5 FepA turn 1 180 SWDPAPL 15 5 FepA turn 2 240 SHFAPHQ 15 5 FepA turn 2 241 SQVPLKS 13 5 FepA turn 2 242 HMSPLGA 12 4 FepA turn 1 181 MHMAPLS 12 4 FepA turn 2 241

a,b,c See footnotes to Table 2.3.

Page 112: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

88

69WTLEP

73 with a score of 17 (Table 2.3, line 3). An additional Ph.D.-12 peptide

was found to align to the core of the FecA Ton box (81

ALTV84

) with a score of 13

within a 5-residue scoring window (Table 2.3, line 9). Two Ph.D.-12 and three

Ph.D.-C7C peptides were determined by RELIC/MATCH to align to FecA

periplasmic turn 8. RELIC/HETEROalign similarity scores that mapped to the

periplasmic surface of FecA indicate that the FecA Ton box region (Figure 2.4B,

region I) is not as highly scored as turn 8 (Figure 2.4B, region II). Furthermore,

RELIC/HETEROalign identified a peptide at turn 6 as being another highly

scored region (yellow), suggesting another possible TonB-binding site undetected

by RELIC/MATCH.

In contrast to FhuA, BtuB, and FecA, RELIC/MATCH did not identify a

cluster of affinity-selected peptides which could align at FepA periplasmic turn 8.

As with the other OM receptors, a peptide cluster aligned to a region proximal to

the Ton box of FepA. In this case, the cluster was comprised of five Ph.D.-12

peptides aligned in a region immediately N-terminal to the FepA Ton box.

Clusters were found to align to FepA periplasmic turns 1 and 2. At FepA turn 1,

two Ph.D.-12 peptides and two Ph.D.-C7C peptides aligned; at FepA turn 2, four

Ph.D.-C7C peptides were found to align (Figure 2.3C). RELIC/HETEROalign

similarity scores that mapped to the periplasm-exposed surface of FepA displayed

a high degree of heterogeneity. However, turns 1 and 2 (Figure 2.4C, regions II

and III, respectively) are prominent as highly-scored regions.

Page 113: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

89

Figure 2.3. Alignments of affinity-selected peptides to the Ton box regions and periplasm-

exposed turns of BtuB, FecA and FepA as identified by RELIC/MATCH. Peptides from the

Ph.D.-12 library are aligned below the protein sequences and peptides from the Ph.D.-C7C library

are aligned above the protein sequences. See Tables 2.3 and 2.4 for respective peptide match

scores and window sizes. Alignment positions are highlighted according to their pairwise

alignment score: +4, black background and white character; +1, grey background and dark

character. A. matches to BtuB Ton box region and to periplasm-exposed turn 8; B. matches to

FecA Ton box region and periplasm-exposed turn 8; C. matches to FepA Ton box region and

periplasm-exposed turns 1 and 2.

A B

C

Page 114: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

90

Figure 2.4. RELIC/HETEROalign similarity scores mapped to the periplasm-exposed surfaces of

BtuB (PDB code 1NQE), FecA (PDB code 1KMO), and FepA (PDB code 1FEP). See legend to

Figure 2.2 for details. The normalized RELIC/HETEROalign similarity scores mapped to the

molecular surfaces shown in A to C are averages of Ph.D-12 and Ph.D.-C7C similarity scores. A.

BtuB; Roman numerals correspond to RELIC/MATCH alignment clusters shown in Figure 2.3A:

(I) BtuB Ton box; (II) BtuB periplasmic turn 8; B. FecA; Roman numerals correspond to

RELIC/MATCH alignment clusters shown in Figure 2.3B: (I) FecA Ton box; (II) FecA

periplasmic turn 8; C. FepA; Roman numerals correspond to RELIC/MATCH alignment clusters

shown in Figure 2.3C: (II) FepA periplasmic turn 1; (III) FepA periplasmic turn 2. The region N-

terminal to FepA Ton box (Figure 2.3C, I) is not visible in 1FEP and therefore not shown.

B A C

Page 115: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

91

2.3.5 Interactions of TonB and [FhuA peptide-MBP] fusion proteins in vitro

Oligonucleotides encoding peptides that are identical to FhuA sequences

within the Ton box/switch helix, cork/β1 strand, and periplasmic turn 8 were

synthesized, annealed, and cloned into a pMal-pIII expression vector. The vector

encodes maltose-binding protein (MBP); FhuA peptide sequences were fused to

the N-terminus of MBP. Peptide fusions corresponded to the following regions in

FhuA: [Ton box-MBP] and [Fhu switch-MBP] both representing region I; [Fhu

cork1-MBP], [Fhu cork2-MBP], and [Fhu cork3-MBP] representing region II; and

[Fhu turn-MBP] representing region III. Amino acid sequences for the FhuA

peptides are listed in Materials and methods. The [FhuA peptide-MBP] fusion

proteins were expressed, purified from E. coli periplasmic extracts, and tested for

their ability to bind to recombinant H6.'TonB in vitro by means of ELISA.

Despite DNA sequence fidelity of cloning and repeated attempts of protein

expression, we were unable to obtain [Fhu cork2-MBP]; the remaining five fusion

proteins were purified to homogeneity in abundant amounts (10 mg l-1

of culture).

H6.'TonB was applied to wells of a Ni-NTA coated microtitre plate. By

tethering the N-terminal hexahistidine tag to the Ni-NTA surface, the C-terminus

of H6.'TonB would be oriented outwards, mimicking the in vivo orientation of

TonB. This strategy markedly increased sensitivity of the assay compared to

randomly coated H6.'TonB (data not shown). Binding of each [FhuA peptide-

MBP] to H6.'TonB was detected (Figure 2.5) in levels greater than those afforded

by wild-type MBP. Binding was specific; incubation of a mixture containing

equimolar amounts ferricrocin-bound FhuA and a given [FhuA peptide-MBP]

Page 116: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

92

reduced ELISA signals compared to incubations with [FhuA peptide-MBP] alone.

A continuous FhuA binding landscape containing the sum of all potential binding

sites was presented to H6.’TonB; fusion proteins displaying FhuA peptide

sequences were effectively out-competed. The level of competition was similar

among all fusion proteins tested and resulted in minimal binding, comparable to

the wild-type MBP control. Equivalent amounts of H6.'TonB were coated per

well; nearly complete competition of all TonB binding sites was achieved. These

data therefore establish that RELIC-identified regions of FhuA interact in vitro

with TonB when displayed as N-terminal peptide fusions to MBP.

Figure 2.5. Interactions of TonB and [FhuA-MBP] fusion proteins in vitro. H6.'TonB (20 pmol)

was incubated with MBP proteins (200 pmol) bearing FhuA sequences as N-terminal fusions:

MBP (wild-type maltose-binding protein); Ton box (6EDTITVTAA

14); Fhu switch

(21

AWGPAAT27

); Fhu cork1 (143

SSPGGLL149

); Fhu cork3 (153

SKRPTTEPL161

); Fhu turn

(583

AKAALSA589

). Numbering is according to the mature FhuA protein sequence. Fusion proteins

bound to H6.'TonB were detected by development of PNPP at 405 nm after incubation with the

appropriate antibodies (see Materials and methods). Black bars indicate signals observed due to

interactions between MBP-fusions and H6.'TonB after 180 min. White bars indicate signals

observed when equimolar amounts of [FhuA peptide–MBP] fusion and FhuA (200 pmol each)

were simultaneously incubated with H6.′TonB for 180 min.

Page 117: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

93

2.4 Discussion

To identify surfaces on bacterial OM receptors in addition to the well-

characterized Ton box that might provide binding sites for TonB, we adopted

experimental strategies of phage display. Peptides were affinity-selected, based

upon their favourable interactions with H6.'TonB. Decreased amino acid

diversity and information contents were used to gauge the statistical merit of our

pool of affinity-selected peptides and indicated enrichment compared to randomly

selected peptides from the parent libraries. Statistical analyses of this pool of

peptides confirmed their affinity towards H6.'TonB; they were not selected by

nonspecific associations.

Using this approach, we identified novel TonB-binding regions on

surfaces of OM receptors that are known to interact with TonB. These regions

localized to periplasm-accessible surfaces. We also identified regions that are

TonB-inaccessible, based upon known crystal structures.

Our use of RELIC/MATCH identified regions on the periplasm-accessible

surfaces of the OM receptors FhuA, BtuB, FecA and FepA with sequence

similarities to the pool of TonB affinity-selected peptides. For the OM receptors

examined, the regions were all similarly located: the Ton box/switch helix region;

the cork domain/1 strand; and one or more periplasmic turns. Because these

three regions do not reveal obvious signature sequences for interactions with

TonB, we conclude that molecular recognition is based upon particular

conformations of peptides within the binding surfaces.

Page 118: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

94

Noteworthy is that the Ton box, a region of known interaction between

TonB and FhuA, was not overly represented among our affinity-selected peptides.

The recent report of Peacock et al. (75) examined interactions between a TonB

construct containing residues 103–239 and synthetic Ton box peptides from

various OM receptors. Isothermal titration calorimetry confirmed weak affinities

between TonB and the Ton box peptides; Kd’s were in the micromolar range.

Complemented by findings that TonB recognizes a conformation of the Ton box

rather than a strict amino acid sequence, this outcome suggests that few phage-

displayed peptides adopted conformations that TonB might recognize. Even if

there were some greater population of phages with sequences similar to the Ton

box regions of OM receptors, their affinities towards TonB might have been too

low and thus may have escaped selection.

Periplasmic turns identified on the OM receptors were significantly

represented in our affinity-selected peptide populations. The Ph.D.-C7C affinity-

selected peptides that mapped to these turns possess conformations, which impose

significant selection constraints for interactions with immobilized TonB. Their

identification is meaningful because they probe regions within TonB that are

similarly constrained. Thus, the periplasmic turns identified in the OM receptors

apparently accommodate bound TonB and represent novel sites of contact beyond

the Ton box. Identification of these regions is consistent with previous findings

of Braun et al. (158), Howard et al.(160), Scott et al (159), and Killman et al.

(161), demonstrating that the FhuA -barrel possesses binding sites necessary to

Page 119: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

95

support TonB-dependent function. OM receptor turns identified in the present

study therefore represent previously inferred TonB binding sites.

The most striking regions within FhuA that were identified by phage

display as interactors with TonB were those of the cork region. Not all identified

regions within the cork are completely exposed to the periplasm, yet when

selected sequences were displayed as [FhuA peptide-MBP] fusions, they were

shown to interact with TonB in vitro. It is difficult to envision TonB’s association

with regions inside the FhuA cork domain, even if the cork were partially

displaced from the barrel. However such regions might represent points of

contact between TonB and FhuA at different stages during siderophore transport.

Hence, our analyses primarily focused on regions clearly exposed to the periplasm

because these regions would most likely represent initial binding sites for TonB.

Our recent biophysical data (77) illustrated TonB’s association with apo-FhuA in

a 2:1 ratio; addition of siderophore enhanced this interaction. We advocated a

model whereby there is an initial encounter complex between FhuA and a

monomer of TonB, followed by a kinetically limiting TonB rearrangement that

facilitates recruitment of a second TonB monomer. Deletion of cork residues 21–

128 of FhuA caused an alteration in the stoichiometry of the complex, as did

deletion of the TonB proline-rich region. In both cases, only 1:1 TonB-FhuA

complexes were observed; however, SPR analysis indicated that these complexes

formed with high affinity. We interpreted these 1:1 complexes as the encounter

complex; multiple sites of interaction are positioned within this complex. Our

phage display data agree with this interpretation because we observe discrete

Page 120: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

96

regions of FhuA that interact with TonB. These results do not exclude aspects of

TonB function that may occur at the CM. The report of Ghosh and Postle (79)

showed that TonB can form dimers that were previously unidentified through in

vitro studies. However, this same report indicated that monomeric TonB

associated with the OM. It is likely that the oligomeric state of TonB is dynamic

throughout the energy transduction cycle; perhaps energy from the proton motive

force unwinds the CM-localized TonB dimer, resulting in a monomeric form of

TonB competent to interact with OM receptors.

Figure 2.6 illustrates a representation of FhuA’s periplasmic surface;

regions that interact with TonB in vitro are highlighted in red. Significantly, the

highlighted regions form a continuous surface to which TonB can bind. For apo-

FhuA (Figure 2.6A), this surface appears segmented; the switch is coiled near turn

8 where its helix conformation is stabilized by hydrophobic interactions. Upon

siderophore binding, the gap between these two surfaces is bridged; the switch

helix unwinds and engages space nearer the cork/1 strand (Figure 2.6B).

Bridging of this space might facilitate stronger interactions of TonB with the cork.

Upon activation via ExbB-ExbD, TonB may then exert additional conformational

changes within the cork that permit passage of the siderophore.

TonB apparently facilitates OM receptor activity in part through its

interaction with the -barrel. Our data are consistent with previous findings (158-

161) because we identified periplasmic turn 8 and the shallow loop leading into

1 strand as binding sites for TonB. Turn 8 forms part of the continuous surface

Page 121: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

97

that bridges the Ton box/switch helix region to the proximal cork regions and it

likely represents one of the initial points of contact with the OM receptor.

To further elucidate molecular mechanism, we examined the electrostatic

surfaces of both the periplasmic faces of FhuA (apo- and ligand-loaded) and the

most recent NMR structure of monomeric TonB. The electrostatic surface of apo-

FhuA contains an electronegative surface near the cork/1 strand that we

identified from phage display as being a potential binding surface for TonB.

Binding of siderophore causes unwinding of the switch helix and presents an

additional electronegative surface to which TonB can bind. This additional

surface is nearly identical to the surface that we show to interact with TonB in

vitro.

Through automated docking of monomeric TonB to the periplasmic face

of FhuA, we obtained an unbiased low energy complex that places TonB directly

over the RELIC-identified surfaces (Figure 2.6C). This surface orients TonB such

that its electropositive Arg-171 fits within the electronegative cleft formed by

residues Glu-159, plus Asp-186 and Asp-187 in FhuA’s periplasmic turn 1

(Figure 2.6D). Significantly, this region was identified by HETEROalign from

affinity-selected peptides in both the Ph.D.-12 and Ph.D.-C7C libraries (Figure

2.2C). In this docked conformation, TonB Gln-160 is oriented toward the lumen

of the FhuA barrel within a space near the switch helix and presumably the Ton

box (Figure 2.6 D). We propose that this TonB–FhuA docking solution may

represent an initial encounter complex between one molecule of TonB and one

molecule of FhuA.

Page 122: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

98

Figure 2.6. TonB-binding surfaces on the periplasmic face of FhuA. Periplasmic faces (green) of

ligand-free FhuA (panel A, PDB code 2FCP) and ligand-loaded FhuA (panel B, 1FCP) are

represented in the same orientation as in Figure 2.2. Indicated in red are the regions shown to

interact in vitro with TonB. Panel C. Docking of monomeric TonB to FhuA: view of the

periplasmic surface of FhuA (see Figure 2.2D for orientation) shaded in green; residues from the

peptides Fhu switch, Fhu cork 1, Fhu cork 2, Fhu cork 3, and Fhu turn shaded in red. The three-

dimensional NMR model of the TonB C-terminal domain (PDB code 1XX3) was tested by the

computer program AUTODOCK (171) for its ability to dock to the periplasmic surface of FhuA

(PDB code 1FCP). Using Lamarckian Genetic Algorithm (LGA) routines (10 LGA runs in total;

population size: 50; max. number of energy evaluations: 250,000), a cluster of three low-energy

docked TonB conformations was obtained (average docking energy 37.63 kcal mol-1

). The lowest

energy (35.7 kcal mol-1

) member of the cluster of docked TonB 151–239 molecules is shown as a

ribbon representation (yellow). Panel D. View of contacts between docked TonB 151–239

(yellow ribbon) and FhuA (green ribbon). TonB residues and labels are shown in blue; FhuA

residues and labels are shown in red. The N-terminus of the FhuA structure (PDB code 1FCP,

residue 19) is indicated by an asterisk.

Page 123: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

99

The recombinant H6.’TonB used in this study is incapable of becoming

energized. However, Howard et al. (160) reported that overexpressed, truncated

TonB constructs exported to the periplasm blocked TonB-dependent processes by

interfering with endogenous TonB function. By associating with OM receptors,

the constructs effectively competed for available TonB binding sites on FhuA and

FecA. They concluded that the interactions were meaningful because the

truncated TonB constructs recognized ligand-loaded receptors despite an inability

to accept energy input from ExbB–ExbD. The interactions were not fortuitous;

they did not occur by non-productive intrinsic affinity between TonB and OM

receptors. Our results are consistent with those of Howard et al., establishing that

we have identified biologically relevant sites of interaction between TonB and

OM receptors extending beyond the Ton box and that include regions of the -

barrel.

In addition to identifying periplasm-accessible TonB binding sites on OM

receptors, we report that our phage display strategies identified distal regions on

OM receptors of potential interaction with TonB. These regions were near the

cork apex and on extracellular loops of FhuA and of other OM receptors (region

II, cork1 designation). Because there are no data demonstrating TonB’s insertion

into the receptor lumen nor interacting with such extracellular regions, we can

only surmise what such interactions might imply. Nevertheless, it is intriguing

that some of our affinity-selected peptides mapped to an apical region of the FhuA

cork domain (Figure 2.1B; 145

PGGLL149

) that structurally overlaps a region on

FepA shown to be important in receptor function (172,173). In FepA, these

Page 124: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

100

residues are located at the extracellular surface of the cork and they cluster near

the barrel wall forming the structurally conserved quadrupole or lock region. By

employing different mutagenesis strategies, Barnard et al. (172) and Chakraborty

et al. (173) demonstrated that mutations of these residues affected ligand

transport, but not ligand binding. An intriguing interpretation of their results is

that this region might somehow be involved in interactions with TonB. Our

affinity-selected peptides point to a similar region within FhuA that is on the

extracellular side of the cork and that clusters near the barrel wall, a region that

we designated cork1. MBP constructs bearing FhuA fusions of this region were

able to bind to TonB in vitro. Using different experimental tools, the studies of

Barnard et al., Chakraborty et al. and our phage display direct attention towards

possible interactions with TonB at regions removed from the periplasmic space.

It is tempting to propose that such a sterically occluded region might interact with

TonB. Cork-barrel interfaces are highly hydrated by water molecules acting as

bridges in a hydrogen bonding network between the two domains (104,151). This

network is similar to those found in transient protein–protein interfaces that

engage in gross domain movements or conformational change. Furthermore, their

analysis identified a region of structural conservation among cork domains of

TonB-dependent OM receptors that they referred to as the latch. The latch

(residues 139–141 in FhuA) was suggested to play a role in the transport process,

perhaps involving an unfolding event during transport. The latch is immediately

adjacent to the cork1 region that we identified in our phage display study and

which we showed to interact with TonB. These concordant observations merit

Page 125: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

101

continuing experimentation to provide additional insights into the molecular

mechanisms of bacterial OM receptors and the role of TonB.

2.5 Acknowledgements

This research was supported by operating grants to J.W.C. from the

Canadian Institutes of Health Research (CIHR) and to L.M. from National

Institutes of Health. J.-N. Gagnon received a PGS-M scholarship and M.D., a

USRA scholarship from Natural Sciences and Engineering Research Council of

Canada. Canada Foundation for Innovation provided infrastructure for molecular

modelling to the Montreal Integrated Genomics Group for Research on Infectious

Pathogens. Sheldon Biotechnology Centre at McGill University is supported by a

Multi-user Maintenance Grant from CIHR. We appreciate contributions of

experimental materials by A. Clements, N.M. Desy, C. Ng; critical reviews of the

manuscript by M. A. Hancock and G. Marczynski; and editorial support by J.A.

Kashul.

Page 126: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

102

Preface to chapter 3

Regions of interaction between TonB and FhuA were predicted by our use

of phage display in chapter 2. Our later crystal structure of TonB bound to FhuA

provided the proof of principle that our strategies are predicting biologically

relevant interactions. In chapter 3, this strategy extends to the prediction and

demonstration that TonB interacts with periplasmic binding protein, FhuD.

Described therein is the use of phage display to predict and localize regions of

interaction between TonB and FhuD. These predictions are then confirmed with

biophysical experiments that revealed the stoichiometry and affinity between

TonB and FhuD. Furthermore, we demonstrated that TonB can interact with the

OM receptor FhuA and FhuD at the same time. We conclude with a model of

how such a ternary complex might facilitate siderophore uptake.

Page 127: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

103

Chapter 3

Interactions between TonB from Escherichia coli and

the periplasmic protein FhuD

David M. Carter, Isabelle R. Miousse, Jean-Nicolas Gagnon, Éric Martinez,

Abigail Clements, Jongchan Lee, Mark A. Hancock, Hubert Gagnon,

Peter D. Pawelek and James W. Coulton

Journal of Biological Chemistry (2006). 281: 35413-35424

Copyright 2006, American Chemical Society

Page 128: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

104

3.0 Summary

For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent

outer membrane receptor of Escherichia coli is required. The periplasmic protein

FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated

permease FhuB/C. We exploited phage display to map protein–protein

interactions in the E. coli cell envelope that contribute to ferrichrome transport.

By panning random phage libraries against TonB and against FhuD, we identified

interaction surfaces on each of these two proteins. Their interactions were

detected in vitro by dynamic light scattering and indicated a 1:1 TonB–FhuD

complex. FhuD residue Thr-181, located within the siderophore binding site and

mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD

T181C was reacted with two thiol-specific fluorescent probes; addition of the

siderophore ferricrocin quenched fluorescence emissions of these conjugates.

Similarly, quenching of fluorescence from both probes confirmed binding of

TonB and established an apparent KD of approximately 300 nM. Prior saturation

of FhuD’s siderophore binding site with ferricrocin did not alter affinity of TonB

for FhuD. Binding, further characterized with surface plasmon resonance,

indicated a higher-affinity complex with KD in the low nanomolar range.

Addition of FhuD to a pre-formed TonB–FhuA complex resulted in formation of

a ternary complex. These observations lead us to propose a novel mechanism in

which TonB acts as a scaffold, directing FhuD to regions within the periplasm

where it is poised to accept and deliver siderophore.

Page 129: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

105

3.1 Introduction

Iron, an essential nutrient for almost all bacterial species, is required for

metabolic processes including electron transfer, oxygen activation and

biosynthesis of amino acids and nucleosides (145). However, Fe3+

is scarce in the

extracellular environment. Gram-negative bacteria have evolved transport

processes that utilize siderophores to scavenge extracellular Fe3+

by high-affinity

chelation. Different siderophore receptors are expressed at the bacterial outer

membrane (OM), each with specificity for a particular metal-chelated

siderophore. Transport of receptor-bound siderophores into the periplasm

requires contribution of energy provided by the TonB–ExbB–ExbD complex that

is anchored in the cytoplasmic membrane (CM). TonB spans the periplasm to

make contacts with cognate OM receptors. By harnessing energy produced from

the proton motive force, TonB may propagate conformational changes to OM

siderophore receptors, resulting in release of siderophore into the periplasm.

The ferrichrome transport system consists of four proteins (FhuA, FhuB,

FhuC, and FhuD) expressed by Gram-negative bacteria. The FhuA protein

comprises two domains: an N-terminal globular cork domain is enclosed by a 22-

stranded C-terminal β-barrel domain (42,150). Connections between β-strands in

the barrel domain are such that long loops participating in ferrichrome binding are

exposed to the extracellular environment; short turns are exposed to the

periplasm. Uptake of ferrichrome is a TonB-dependent process, mediated by

contacts between TonB and the OM receptor FhuA. TonB is elongated and has

three domains: an N-terminal domain anchored in the CM, an intermediate

Page 130: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

106

domain containing Pro-Glu and Pro-Lys repeats, and a globular C-terminal

domain with a central β-sheet and two α-helices. To date, structural data is only

available for the C-terminal domain (72,74,75). We recently solved the crystal

structure of the 1:1 TonB–FhuA complex (96). The C-terminal domain of TonB

makes extensive contacts with the N-terminal consensus Ton box of FhuA, as

well as residues Ala 26 and Glu 56 of the cork domain and with periplasmic turns

8 and 10. These contacts orient TonB such that it may mediate conformational

disruption of the internal cork domain of FhuA, allowing for passage of

siderophore into the periplasm.

Although recent structural and biophysical data have clarified initial steps

of the siderophore transport cycle involving TonB–receptor interactions, little is

known about the fate of the siderophore once transported into the periplasm.

Specifically, the molecular mechanisms of siderophore transport from periplasm

to cytoplasm a largely uncharacterized. FhuD in the periplasm binds

hydroxamate siderophores ferrichrome, coprogen and aerobactin (174). Loss of

FhuD function in vivo prevented growth of E. coli under iron-limiting conditions

when ferrichrome, coprogen, or aerobactin were used as the sole iron source,

suggesting that FhuD is a necessary component of the hydroxamate siderophore

transport system (174). FhuD was reported to interact with regions of the CM-

embedded permease FhuB. Interactions between FhuD and FhuB have been

demonstrated by cross-linking studies, protease protection assays (113), and

ELISA (132). Interaction of FhuB with FhuD is apparently independent of

siderophore binding by FhuD (113). Taken together, these results suggest that

Page 131: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

107

FhuD functions as a carrier protein: ferrichrome released from the OM receptor is

delivered by FhuD to the permease. The integral membrane protein FhuB then

translocates the siderophore into the cytoplasm mediated by ATP hydrolysis of

FhuC (175).

The crystal structure of FhuD in complex with gallichrome, a ferrichrome

analogue, has been reported (111), as well as structures of FhuD in complex with

albomycin, coprogen, and Desferal (112). The fold of this 32 kDa protein is

bilobal: globular N- and C-terminal domains are connected by a long α-helix that

confers rigidity to the protein. The siderophore binding site residing in a shallow

cleft between the two lobes is hydrophobic, having predominantly aromatic

residues. Siderophore binds to FhuD through both hydrophobic and hydrophilic

interactions. Methylene carbon atoms in the siderophore form hydrophobic

interactions with numerous aromatic FhuD residues in the binding cleft.

Hydrogen bonds are formed between hydroxamate groups of the siderophore and

FhuD residues Arg-84 and Tyr-106. A hydrogen bond with the siderophore is

also formed with FhuD residues Asn-215 and Ser-219 through an intermediate

water molecule.

The overall fold of FhuD is similar to that of BtuF (114,120), the

periplasmic cobalamin-binding protein of E. coli. Periplasmic metal-binding

proteins TroA (176) and PsaA (177) are also structurally related to FhuD. These

proteins share a fold distinct from those of classical periplasmic proteins such as

maltose binding protein (178). However unlike maltose binding protein, FhuD

does not exhibit gross conformational rearrangements upon ligand binding. The

Page 132: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

108

linker connecting the N-terminal and C-terminal domains in FhuD is a kinked α-

helix that crosses these domains only once. The structure of FhuD and the

hydrophobicity of the siderophore binding site suggest that large-scale opening

and closing of the binding site does not occur upon siderophore binding and

release (178). Molecular dynamics simulations also suggest that FhuD is

conformationally rigid, but that subtle conformational differences in the C-

terminal domain between the apo- and holo-forms may be sufficient for

discrimination by FhuB (119).

What molecular events result in capture of ferrichrome by FhuD following

its TonB-dependent release from FhuA? Given the apparent weak affinity (1 M)

of ferrichrome for FhuD (113), binding is unlikely to be a diffusion-governed

process. Efficiency of siderophore capture would be enhanced by positioning a

binding protein proximal to the lumen of the OM receptor. This organization

would promote direct transfer of ferrichrome from FhuA to FhuD. Here we report

the first biophysical evidence that TonB specifically interacts with FhuD.

Discrete regions of protein–protein interactions on the surfaces of both FhuD and

TonB were identified by phage display. Interactions were confirmed by dynamic

light scattering, fluorescence spectroscopy and surface plasmon resonance. Our

results suggest that siderophore released from FhuA during the transport cycle is

transferred to FhuD via a coordinated transfer mechanism mediated by TonB.

Hence, TonB would act as a periplasm-spanning scaffold, directly connecting

siderophore transport events between the OM and CM.

Page 133: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

109

3.2 Materials and methods

3.2.1 Bacterial strains, phage libraries, and media

Random peptide phage libraries Ph.D.-C7C and Ph.D.-12 were purchased

from New England Biolabs (NEB); E. coli ER2738 also from NEB was used for

amplification and titration of phage M13 pools. E. coli ER2566 was used to

express recombinant TonBs (77); E. coli BL21 (DE3) pLysS was used to express

recombinant FhuDs. Plasmid pCMK01 expresses a hexahistidine-tagged TonB

32-239 (25 kDa; hereafter identified as TonB) and pWA01 expresses a

hexahistidine-tagged TonB 32-239 with an engineered cysteine residue at its

amino terminus (hereafter identified as Cys-TonB) (94,95). FhuD was expressed

from pMR21 provided by W. Köster (VIDO, Saskatoon, SK); the N-terminus of

FhuD containing the signal sequence was removed and replaced by a

decahistidine tag (179) (32 kDa; hereafter identified as FhuD). Plasmid pMR21

was commercially mutated to cysteine at Thr181 by Norclone Biotech

Laboratories (London, ON); this protein is hereafter identified as FhuD T181C.

Mutagenesis was confirmed by DNA sequencing at Sheldon Biotechnology

Centre, McGill University (Montreal, QC). All bacteria were cultured in Luria

Bertani (LB) broth containing antibiotics when necessary.

3.2.2 Chemicals and reagents

5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and

isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Biovectra

Page 134: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

110

(Charlottetown, PE). Protein-grade Tween-20 was purchased from Calbiochem.

Antibiotics were purchased from Sigma-Aldrich. Ni-NTA resin used for protein

purifications was purchased from Qiagen. The reducing agent Tris-(2-

carboxyethyl)phosphine (TCEP) and fluorescent dyes 5-((((2-

iodoacetyl)amino)ethyl)amino)naphthaline-1-sulfonic acid (AEDANS) and 7-

diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) were

purchased from Invitrogen.

3.2.3 Protein purification

TonB and Cys-TonB were purified as described previously (77). To

purify overexpressed FhuD or FhuD T181C, cell pellets were suspended in 50 ml

of buffer A containing 50 mM Tris pH 8.2, 150 mM NaCl and 5 mM imidazole

plus one Complete mini EDTA-free protease inhibitor cocktail tablet (Roche);

0.16 mg/ml lysozyme and 16 µM phenylmethyl sulphonylfluoride (PMSF) were

then added. Cells were shaken at room temperature for 30 min, followed by

addition of 0.04 mg/ml DNase, 0.04 mg/ml RNase, and an additional 16 µM

PMSF. To lyse bacteria, cells were passed twice through an Emulsiflex-C5

(Avestin, Ottawa, ON). Cell lysate was centrifuged (27,000 × g, 4 °C) for 50 min

and filtered through 0.45 m syringe filters. Filtered cell extracts containing

FhuD or FhuD T181C were applied to Ni-NTA resin equilibrated with buffer A.

FhuDs were eluted with 50 mM Tris pH 8.2 containing 125 mM imidazole,

pooled and applied to a POROS HQ 20 anion exchange column (Applied

Biosystems). Bound proteins were washed with 50 mM Tris pH 8.2 containing

Page 135: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

111

125 mM imidazole, eluted with 160 mM NaCl and applied inline to a POROS MC

20 column (Applied Biosystems). After extensive washing, proteins were eluted

with 50 mM Tris pH 8.2 and 120 mM imidazole and applied to a second POROS

HQ 20 column, washed as described above, and eluted with 180 mM NaCl in 50

mM Tris pH 8.2. Purified proteins were dialyzed in a 24,000 Mr cut-off dialysis

membrane (SpectraPor) for 16 h at 4 °C in 100 mM Hepes, 150 mM NaCl, pH

7.4. Homogeneity of FhuDs was confirmed by SDS-PAGE and silver staining of

750 ng of total protein. Concentrations of protein were determined by either a

Bradford or BCA assay using bovine serum albumin as standard.

3.2.4 Phage display

Phage panning against TonB as target was described previously (180).

Purified FhuD was diluted to 100 g/ml in TBS (Tris-buffered saline: 50 mM

Tris, pH 7.5, 150 mM NaCl) and 150 l of protein was adsorbed to a polystyrene

microtitre plate (Nunc Maxisorp). Plates coated with immobilized FhuD were

incubated for 16 h at 4 °C followed by blocking (2 h at 37 °C) with TBS

containing 5 mg/ml bovine serum albumin. The unselected phage library (NEB)

was then added. Phage panning, clone isolation, DNA sequencing, and

bioinformatic analyses were performed as described previously (180).

3.2.5 Dynamic light scattering

Light scattering was measured from purified TonB and FhuD dialyzed

twice (18 h, 4 °C) in 100 mM Hepes, pH 7.4 containing 150 mM NaCl. Purified

Page 136: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

112

[Fhu switch-MBP] fusion protein (180) (containing FhuA residues

21AWGPAAT

27 fused to the N-terminus of maltose binding protein) and BSA

were dialyzed against the same buffer and used in DLS measurements as positive

and negative controls, respectively. TonB (4.0 M) and FhuD (3.0 M) were

separately analyzed as discrete scattering species. Similarly, BSA (1.5 µM) and

[Fhu switch-MBP] (2.5 µM) were analyzed separately.

For a 1:1 molar ratio of TonB to FhuD, each protein at 1.7 µM was mixed

prior to recording DLS readings. For 1:1 mixtures of TonB with BSA or with

[Fhu switch-MBP], proteins were each at 1.0 µM. Protein mixtures were

incubated for 30 min at room temperature prior to centrifugation and analysis.

Data acquisition was performed in a 12 µl quartz cuvette at 20 °C using a

temperature controlled DynaPro E-50-830 dynamic light scattering instrument

(Protein Solutions, Charlottesville, VA). The scattering signal was measured at a

wavelength of 824.9 nm and an angle of 90°. Data were collected for 7000 s with

a 10 s averaging time, and replicated with two independent protein preparations.

From the Dynamics v.6.3.18 software (Protein Solutions, Charlottesville, VA),

data were filtered (baseline < 1.01 and sum of squares < 500) before exporting to

Sedfit v.9.3(143). Analyses of hydrodynamic radii (Rh) were performed using the

continuous intensity distribution model (143) in Sedfit at a resolution of 100 for

radii between 1 and 50 nm. Buffer densities and viscosities were set to 1.00442

and 0.01065, respectively, as determined by Sednterp v.1.08

(www.jphilo.mailway.com). All values of Rh from the Dynamics software

Page 137: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

113

exhibited less than 14% polydispersity, except for the TonB–FhuD mixture and

[Fhu switch-MBP] (21% and 19%, respectively).

For Sedfit analyses of Discrete Non-interacting Species (DNS),

autocorrelation data sets were imported from the Dynamics software package and

fit to a single species field autocorrelation function. Values of s for TonB were

previously determined (77). Using analytical ultracentrifugation, we determined

by sedimentation velocity experiments sedimentation coefficients for FhuD and

the TonB–FhuD complex: 2.27 s and 3.5 s, respectively. From literature reports, s

values for MBP (181) and BSA (182) were obtained. All were constrained in

DNS analyses. Molecular mass values for discrete scattering species, either

uncomplexed TonB, uncomplexed FhuD, or 1:1 heterocomplexes were initially

set to predicted values and then refined by nonlinear regression until rmsd errors

were minimized. In addition to proteins TonB, FhuD, [Fhu switch-MBP] or

complexes formed by these proteins, two scattering species were observed; the

Dynamics program predicted these uncharacterized species to have hydrodynamic

radii of ~1 nm and ~100 nm respectively. Hydrodynamic parameters for these

species were factored into DLS analyses to optimize fits to the autocorrelation

function.

3.2.6 Fluorescence spectroscopy

The fluorescent dye AEDANS was conjugated to FhuD and FhuD T181C

in a reaction buffer of 100 mM Hepes pH 7.4, 150 mM NaCl. Following

reduction of disulfide bonds with a ten-fold molar excess of TCEP, dye was added

Page 138: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

114

to a ten-fold molar excess. Conjugation proceeded in the dark with stirring for 4 h

at room temperature. Reactions were quenched by addition of β-mercaptoethanol.

Excess label was removed by exhaustive dialysis against four 1 l changes of 100

mM Hepes, pH 7.4 containing 150 mM NaCl in the dark at 4 °C. After dialysis,

free dye was present at picomolar concentrations. Conjugates were then

centrifuged at 18,000 × g for 30 min at 4 °C. Labeled proteins were stored at 4 °C

in the dark. Conjugation of FhuD T181C with the dye MDCC was performed as

described above except that MDCC was dissolved in DMSO prior to its addition

to protein. Efficiency of labeling (mol dye:mol protein) was calculated from

absorption data using the following tabulated (Invitrogen) molar extinction

coefficients: 5700 M–1

cm–1

at 336 nm for AEDANS and 50,000 M–1

cm–1

at 419

nm for MDCC and from protein concentrations as determined by protein assays.

Fluorescence data were collected with a Varian Cary Eclipse fluorescence

spectrophotometer. Emission spectra were recorded at excitation and emission

wavelengths of 280 nm and 340 nm respectively for intrinsic fluorescence

measurements; at 336 nm and 490 nm respectively for AEDANS-labeled FhuD

and FhuD T181C; and at 419 nm and 466 nm respectively for MDCC-labeled

FhuD T181C. Excitation and emission slits were set between 2.5−5 nm and 5−10

nm respectively. Measurements were taken in triplicate at 20 °C. Data were

corrected for changes in fluorescence intensity attributed to dilution of protein and

the minimal fluorescence contributions of Fcn, TonB and buffer (100 mM Hepes

pH 7.4, 150 mM NaCl).

Page 139: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

115

Binding of Fcn to either FhuD (1.5 M) or FhuD T181C (0.5 M) was

monitored by recording the fluorescence emission after additions of Fcn up to a

ten-fold molar excess. For each data point, Fcn was added from a stock solution

and after three minute incubation, the change in fluorescence was recorded.

Titrations of labeled conjugates with either Fcn or TonB were conducted in an

identical manner. Fluorescence quenching was expressed as the percentage

decrease in fluorescence upon ligand addition compared to the theoretical

maximum whereby quenching would result in complete loss of fluorescence.

Data were fit (Sigmaplot) to an equation describing a rectangular hyperbola using

the single binding site model or to a sum of two hyperbolics using the model that

describes two independent binding sites.

3.2.7 Surface plasmon resonance (SPR)

Binding interactions between TonB and FhuD or between Cys-TonB and

FhuD were examined in real-time using BIACORE 2000/3000 instrumentation

with research-grade CM4 sensor chips (BiacoreAB, Uppsala, Sweden).

Experiments were performed in triplicate at 25 oC using filtered (0.2 m) and

degassed HBS-ET (50 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05%

(v/v) Tween-20). EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbo diimide), NHS

(N-hydroxysuccinimide), and PDEA (2-(2-pyridinyldithio)ethaneamine) were

from BiacoreAB. Protein-grade detergents (10% Tween-20, 10% Triton X-100,

30% Empigen) were from Calbiochem. All other chemicals were reagent grade

quality.

Page 140: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

116

For amine coupling, TonB was immobilized according to a standard

Biacore protocol. For ligand thiol-coupling, 20 l of freshly mixed solution I

(200 mM EDC and 50 mM NHS in water) was injected (5l/min) over the sensor

chip activating carboxymethyl groups to reactive esters. Reactive thiol groups

were then introduced by a 30 l injection of freshly prepared solution II (80 mM

PDEA in 0.1 M sodium borate pH 8.5). Diluted Cys-TonB ligand (3 g/ml in 10

mM sodium acetate pH 4.5) was injected manually until ~120 RU were bound.

Finally, three injections (20 l) of freshly prepared solution III (50 mM L-cysteine

in 0.1 M sodium formate pH 4.3 containing 1 M NaCl) deactivated excess

reactive groups and removed any non-specifically bound ligand. Coupling

efficiencies were typically ~50%. Reference surfaces were prepared in a similar

manner without any ligand addition.

Immobilized TonB and Cys-TonB surfaces were washed overnight at 5

l/min in running buffer. Prior to use, FhuD analyte was dialyzed against HBS-

ET and immobilized TonB or Cys-TonB surfaces were conditioned at 50 l/min

using regeneration scheme A as follows: two 25 l injections each of (i) 0.05%

(v/v) Empigen, 0.5 M NaCl, 50 mM EDTA, 10 mM NaOH in HBS-ET, (ii) 0.05%

(v/v) Triton X-100, 0.5 M NaCl, 50 mM EDTA, 10 mM NaOH in HBS-ET, and

(iii) HBS-ET. For kinetic experiments, FhuD (0.1-1 M in the absence and

presence of a ten-fold molar excess of Fcn) was injected at 50 l/min (120 s

association + 120 s dissociation) over amine-coupled TonB or thiol-coupled Cys-

TonB. Surface performance and mass transfer tests confirmed that the ligand

density and regeneration conditions were appropriate. All acquired data were

Page 141: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

117

double-referenced (183) and analyzed globally according to the simple 1:1

binding model (A + B = AB) or to the heterogeneous ligand model in the

BIAevaluation 4.1 software (BiacoreAB). Kinetic estimates represent fits to the

experimental data with χ2 values below 1.

Multi-component SPR analyses between FhuA, TonB and FhuD were

performed. Initially, amine-coupled TonB surfaces (250 RU) or thiol-coupled

Cys-TonB surfaces (100 RU) were prepared. Then, either a TonB–FhuA or a

TonB–FhuD binary complex was formed by injecting each analyte at 50 l/min.

By injecting FhuD over TonB–FhuA complexes or by injecting FhuA over TonB–

FhuD complexes, ternary complex formation was assessed.

3.3 Results

3.3.1 Identification of TonB-binding sites on FhuD by phage display

Following affinity selection versus immobilized TonB (180), 135 unique

disulfide-constrained peptides from the Ph.D.-C7C library and 105 unique linear

peptides from the Ph.D.-12 library were analyzed. These phage-displayed

peptides were scanned for their similarity to the primary sequence of FhuD using

the REceptor LIgand Contacts (RELIC) program RELIC/MATCH (168). Among

these sequences, 8 from the Ph.D.-C7C library (Table 3.1) and 13 from the Ph.D.-

12 library (Table 3.2) were found to share similarities with the primary sequence

of FhuD. The Ph.D.-C7C and Ph.D.-12 sequences were observed (Figure 3.1) to

cluster at four discrete regions along FhuD: loop 2 (region I), helix 2 (region II),

Page 142: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

118

loop 8 (region III), and loop 23 (region IV). When these four regions were

mapped (Figure 3.2A) onto the surface of the three-dimensional structure of FhuD

(PDB code: 1EFD), they displayed a binding surface that overlaps the siderophore

binding site (Figure 3.2B). Regions I, II, and IV comprise a continuous binding

surface of approximately 17 Å x 17 Å that is formed at the base of the siderophore

binding pocket. In addition to loop 23, region IV also includes residues from

helix 13 near the C-terminus of FhuD. Although the surface-exposed residues in

region III could form a potential TonB-binding landscape with regions I, II, and

IV, residues 81-88 in loop 3 preclude formation of such a continuum. No FhuD

residues within this interval were identified by our phage display analysis,

suggesting that loop 3 is not TonB-binding. Were there a continuous landscape of

all four regions, it would require a displacement of FhuD’s loop 3 to

accommodate TonB binding. Recent molecular dynamics simulations on the

FhuD structure (119) indicated that the C-terminal domain of FhuD has more

overall mobility than the N-terminal domain. However, within the relatively

static N-terminal domain, loop 3 was observed to be the most mobile region.

3.3.2 Identification of FhuD-binding sites on TonB by phage display

Purified FhuD was immobilized and sequentially panned with the Ph.D.-

C7C library and with the Ph.D.-12 library. Panning yielded 109 unique sequences

from the Ph.D.-C7C library and 38 unique sequences from the Ph.D.-12 library.

Of these sequences, 15 from the Ph.D.-C7C library (Table 3.3) and 10 from the

Ph.D.-12 library (Table 3.4) were found by RELIC/MATCH to be similar to the

Page 143: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

119

primary sequence of TonB. When aligned to TonB, these sequences were

observed (Figure 3.3) to cluster at three discrete TonB regions: an N-terminal

domain (region I), an intermediate domain (region II), and the C-terminal domain

(region III). Three-dimensional structural information has been reported

(72,74,75) only for the C-terminal domain of TonB. We mapped (Figure 3.4A)

the cluster from region III to the NMR structure of the TonB C-terminal domain

(PDB code: 1XX3). Region III forms a continuous solvent-exposed binding

surface on TonB (Figure 3.4B), adjacent to FhuA-binding residues that we

recently observed in the TonB–FhuA crystal structure (96). These observations

suggest a potential FhuA–TonB–FhuD ternary complex. Since the three-

dimensional structure of TonB residues in the N-terminal domain and

intermediate domains are unknown, we consider the possibility that residues from

TonB regions I and II could form a continuous FhuD-binding landscape with

TonB region III.

Page 144: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

120

Table 3.1. RELIC/MATCH identification of TonB-affinity-selected Ph.D.-C7C peptides

corresponding to FhuD sequences.

Peptidea

Peptide match

scoreb

Scoring

window

(residues)

Region Alignment

positionc

PYGAALH 16 5 loop 2 26 PYGAALH 16 5 loop 23 245 YGGATLL 15 5 loop 23 245 QPAVANT 13 5 loop 2 28 SYLNVMH 13 5 loop 23 249 TGPLPNR 13 4 helix 2 43 NPTPEKR 13 4 loop 8 78 KPSSPPF 12 4 helix 2 40

aResidues contained in Scoring window are shown in bold.

bSum of pairwise comparisons of aligned residues within the Scoring Window.

cPosition of first residue of the Scoring window numbering from the N-terminus of the

mature protein.

Table 3.2. RELIC/MATCH identification of TonB-affinity-selected Ph.D.-12 peptides

corresponding to FhuD sequences.

Peptidea

Peptide match

scoreb

Scoring

window

(residues)

Region Alignment

positionc

LLADTTHHRPWT 17 7 loop 2 30 HWKHPWGAWDTL 16 7 loop 2 26 KVWSLEPPGPAA 15 5 helix 2 41 YSPPSPEPPRIK 15 5 loop 8 78 QDRGILVEPPRM 14 8 helix 2 36 DFDVSFLSARMR 14 8 loop 23 244 KLWELNPPQVRT 14 7 helix 2 37 SPAPTNNYTYRL 14 6 loop 2 30 TQPLGLLPSRHL 14 5 loop 2 22 QTALITIHHSLT 13 6 loop 2 32 YGNSLPPRLGPP 13 5 loop 23 245 LWAKLWVVPERA 12 5 helix 2 36 SANLSWRESWPT 12 5 loop 23 246

a,b,c See footnotes to Table 3.1.

Page 145: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

121

Figure 3.1. Alignments of TonB affinity-selected peptides to FhuD as identified by

RELIC/MATCH. A. Region I: FhuD loop 2; B. Region II: FhuD helix 2; C. Region III: FhuD

loop 8; D. Region IV: FhuD loop 23. Peptides from the Ph.D.-12 library are aligned below the

FhuD sequence; peptides from the Ph.D.-C7C library are aligned above the FhuD sequence. See

Tables 3.1 and 3.2 for peptide match scores and window sizes. Alignment positions are

highlighted according to their pairwise alignment score: +4, black background and white

character; +1, grey background and dark character.

Page 146: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

122

Figure 3.2. TonB-binding regions identified by phage display mapped to FhuD (PDB code

1EFD). A. Ribbon representation of FhuD (blue) with predicted TonB-binding regions shaded

yellow. Regions corresponding to RELIC/MATCH alignment clusters shown in Figure 3.1 are

indicated by Roman numerals: (I) loop 2; (II) helix 2; (III) loop 8; (IV) loop 23; B. molecular

surface representation of FhuD (blue) with predicted TonB-binding regions shaded yellow. The

bound ligand gallichrome from the 1EFD structure is shown in stick representation and colored by

atoms (carbon: white, nitrogen: blue, oxygen: red).

Page 147: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

123

Table 3.3. RELIC/MATCH identification of FhuD-affinity-selected Ph.D.-C7C peptides

corresponding to TonB sequences.

Peptidea

Peptide match

scoreb

Scoring

window

(residues)

Region Alignment

positionc

PAPERPQ 16 6 N-term 39 HASPAHN 15 6 intermediate 121 VISAASQ 15 6 C-term 146 QSFPRQL 14 6 C-term 149 NRPSSWL 14 5 intermediate 119 TAENSSP 13 5 intermediate 124 KTSPAWI 13 5 intermediate 127 MTARTTS 13 5 intermediate 129 ISPAQSS 13 4 N-term 39 PAVPAKA 12 5 N-term 36 HLAPAAR 12 5 intermediate 127 KALMRTS 12 5 C-term 153 KPLFHNT 12 4 intermediate 122 HHWAPTR 12 4 intermediate 126 HNMPAQT 12 3 N-term 38

a,b,c See footnotes to Table 3.1.

Table 3.4. RELIC/MATCH identification of FhuD-affinity-selected Ph.D.-12 peptides

corresponding to TonB sequences.

Peptidea

Peptide match

scoreb

Scoring

window

(residues)

Region Alignment

positionc

LHTPWHLPAPEI 16 4 N-term 32 KSLSRHDHIHHH 15 6 C-term 153 YHSPPHTPPAPL 14 6 intermediate 122 SFVGLVELPQNL 14 5 N-term 31 VSRHQSWHPHDL 14 5 C-term 155 KTLTLPLSNTSK 13 6 intermediate 119 KIMRMPRLMTRN 13 6 C-term 147 LHFPLDYPQALG 13 5 C-term 145 WHSPWSTPPAPS 13 4 N-term 31 LHWPLYTPPASP 12 4 N-term 33

a,b,c See footnotes to Table 3.1.

Page 148: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

124

Figure 3.3. Alignments of FhuD affinity-selected peptides to TonB. A. Region I: TonB N-

terminal domain; B. Region II: TonB intermediate domain; C. Region III: TonB C-terminal

domain. Peptides from the Ph.D.-12 library are aligned below the TonB sequence; peptides from

the Ph.D.-C7C library are aligned above the TonB sequence. See Tables 3.3 and 3.4 for peptide

match scores and window sizes. Alignment positions are highlighted according to their pairwise

alignment score: +4, black background and white character; +1, grey background and dark

character.

Page 149: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

125

Figure 3.4. FhuD-binding region identified by phage display mapped to TonB (PDB code 1XX3).

A. Ribbon representation of the TonB C-terminal domain (yellow) with predicted FhuD-binding

region III shaded blue. The region corresponding to RELIC/MATCH alignment cluster shown in

Figure 3.3C is indicated by the Roman numeral (III); B. molecular surface representation of TonB

(yellow) with the predicted FhuD-binding region III shaded blue.

Page 150: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

126

3.3.3 Detection of a TonB–FhuD complex by dynamic light scattering

To identify and characterize a TonB–FhuD complex, we employed

dynamic light scattering, a technique previously shown (184) to be effective for

analyzing protein–protein complexes. Analysis of hydrodynamic distribution

with the program Sedfit (143) revealed discrete hydrodynamic radii (Table 3.5)

for all proteins, each with rmsd values less than 0.0097. Given its larger frictional

ratio (77), TonB would exhibit a larger hydrodynamic radius (Rh) despite a lower

molecular mass compared to FhuD. Hence, TonB and FhuD exhibited similar Rh

values. The Rh obtained from an equimolar mixture of these proteins indicated

that a 1:1 heterocomplex had formed. As a control experiment, we observed an

increase in Rh when a [Fhu switch-MBP] fusion protein harbouring a previously

characterized (180) TonB-binding peptide was mixed with TonB, indicating

formation of a TonB–[Fhu switch-MBP] heterocomplex. No change in Rh was

observed after mixing TonB with BSA, as compared to the Rh for each individual

protein, indicating no formation of a TonB–BSA complex (data not shown).

aRh, hydrodynamic radius calculated from continuous intensity distribution model in

Sedfit v.9.3. bRmsd, root mean square deviation from best fits to the autocorrelation curve for Rh

analyses. cPercent mass from DNS analyses, molecular mass >1000 kDa accounted for less than

11% and the remainder was comprised of molecular mass <1.2 kDa. dRmsd for

discrete non interacting species analysis.

Table 3.5. DLS analysis of TonB, FhuD and MBP-switch fusion.

Page 151: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

127

To estimate the molecular mass of a TonB–FhuD heterocomplex, a

Discrete Non-interacting Species (DNS) analysis was performed using Sedfit.

Results from DNS analyses clearly indicated formation of a 1:1 TonB–FhuD

heterocomplex. Molecular mass values for uncomplexed TonB and uncomplexed

FhuD determined by Sedfit from DLS autocorrelation data agree with their

predicted values (Table 3.5, rows 1 and 2). A 1:1 mixture of TonB and FhuD

resulted in formation of a scattering species with a refined molecular mass of 58

kDa (Table 3.5, row 3), corresponding to a 1:1 TonB–FhuD complex.

Both our phage display outcomes (180) and the X-ray crystallographic

structure (96) of the TonB–FhuA complex demonstrated that residues in the

switch helix region of FhuA interact directly with TonB. DLS analyses of [Fhu

switch-MBP] fusion protein uncomplexed or in complex with TonB yielded

results (Table 3.5, rows 4, 5) in agreement with these previous analyses. The

refined molecular mass for uncomplexed [Fhu switch-MBP] agreed with its

predicted molecular mass. When [Fhu switch-MBP] was mixed with TonB in a

1:1 molar ratio, an abundant scattering species of approximately 61 kDa was

observed, consistent with formation of a 1:1 heterocomplex between TonB and

the fusion protein.

3.3.4 Detection of a TonB–FhuD complex by fluorescence spectroscopy

Guided by the TonB-binding surface on FhuD that was predicted from

phage display, we generated a mutant, FhuD T181C, to which were conjugated

thiol-reactive probes capable of reporting changes in local environment. This

Page 152: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

128

mutant was assessed for its binding of ligand and for its binding of TonB.

Rohrbach et al. (113) observed that addition of ferrichrome caused marked

quenching of FhuD’s intrinsic fluorescence and used this observation to quantify

binding of various ligands. We extended this feature to measure the ligand

binding capacity of FhuD T181C. Addition of Fcn to either FhuD or to FhuD

T181C caused substantial decreases in emission maxima. Binding curves were

generated by plotting the percentage decrease in fluorescence as a function of Fcn

added (Figure 3.5A). Fits of these data (Table 3.6) to a single binding site model

yielded similar apparent dissociation constants (KD app) of either 1.2 ± 0.2 M

(FhuD) or 0.6 ± 0.2 M (FhuD T181C), in agreement with the previously reported

(113) KD of 1 M. Our results establish that the T181C mutation does not

compromise ligand binding.

Taking advantage of the environmental sensitivity of the thiol-reactive

probes AEDANS and MDCC, each probe was conjugated to FhuD T181C.

Absorbance data (not shown) indicated that AEDANS labeled FhuD T181C at

approximately a 1:1 ratio, consistent with Cys 181 being solvent accessible and

reactive. MDCC labeled approximately 1 mole of conjugate for every 3 mole of

protein. This outcome likely resulted from labeling conditions. MDCC must be

dissolved in DMSO; addition of this labeling solution to FhuD resulted in slight

precipitation compared to the aqueous labeling conditions used with AEDANS, in

which no precipitation was observed.

Page 153: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

129

Figure 3.5. Binding of Fcn to FhuD and to FhuD T181C. A. FhuD (●) and FhuD T181C (■) were titrated with the indicated amounts of Fcn; quenching of intrinsic fluorescence is plotted as a function of Fcn concentration; B. binding of Fcn to AEDANS-labeled FhuD T181C

(●) and to MDCC-labeled FhuD T181C (▼). Proteins were titrated with Fcn and quenching of

probe fluorescence was plotted as a function of Fcn added. Error bars in panels A and B represent

the standard deviation from three independent experiments. Lines through data indicate best fits to

a single binding site model as determined with Sigmaplot.

Upon addition of Fcn to AEDANS-labeled or to MDCC-labeled FhuD

T181C, extrinsic fluorescence was quenched. Titration of these conjugates with

Fcn is represented by the binding curves depicted in Figure 3.5B. Fits of these

data (Table 3.6) to a single binding site model yielded a similar KD app: 0.9 ± 0.2

M for AEDANS-labeled FhuD T181C; 0.31± 0.03 M for MDCC-labeled FhuD

T181C. These binding constants agree with those determined by intrinsic protein

fluorescence and thus validate the utility of our experimental approach.

Page 154: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

130

Quenching of each probe’s fluorescence upon addition of Fcn therefore reported

occupancy of the FhuD siderophore binding site.

FhuD T181C labeled with either AEDANS or MDCC demonstrated

marked changes in fluorescence when mixed with TonB. Upon addition of TonB

to labeled conjugates, quenching was observed (Figure 3.6), demonstrating that

presence of TonB altered the probe’s environment. These changes were not

observed when labeled FhuD T181C was mixed with an excess of BSA (data not

shown). At low micromolar amounts of added TonB, saturation was achieved.

Fits of these data to a single binding site model generated values listed in Table

3.6. The KD app for the TonB–FhuD complex was 0.31 ± 0.05 M for AEDANS-

labeled FhuD T181C and 0.27 ± 0.04 M for MDCC-labeled FhuD T181C. Prior

saturation of the FhuD binding site with Fcn did not affect binding of TonB

(Figure 3.6) because similar dissociation constants (Table 3.6) were determined

despite occupancy.

a Given by the equation y = Bmax × [L]/(Kd+[L]), where [L] indicates ligand

concentration; reported uncertainties represent standard errors associated with best

fits to the single binding site model. b Fcn, ferricrocin.

Table 3.6. Summary of ligand binding parameters fit to a single site saturation ligand

binding modela

Page 155: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

131

Figure 3.6. Binding of TonB to AEDANS-labeled FhuD T181C and to MDCC-labeled FhuD

T181C. TonB was added to a solution of labeled FhuD (in the absence or presence of Fcn) and

changes in extrinsic fluorescence were recorded. A. Response upon addition of TonB to either

FhuD T181C-AEDANS (●) or Fcn-bound FhuD T181C-AEDANS (▼); B. response upon

addition of TonB to either FhuD T181C-MDCC (●) or Fcn-bound FhuD T181C-MDCC (▼). Lines through data indicate best fits to a single binding site model as determined with Sigmaplot.

Results are representative of three experiments.

Given the spatial separation of putative interaction surfaces on TonB and

FhuD, we attempted fits of our fluorescence data to a model describing two

independent binding sites. When the data from titration of AEDANS-labeled

FhuD T181C with TonB were fit to this model, a larger standard error was

obtained and the resulting KD app values were not meaningful (data not shown).

Conversely, when the data from titration of MDCC-labeled FhuD T181C with

TonB were fit to this model, the standard error improved compared to the single

binding site model. However, the resulting KD app values obtained from this

Page 156: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

132

procedure yielded large uncertainties confounding their interpretation. While we

cannot discount the possibility of two binding sites for TonB on FhuD, our results

favour a single binding site.

Alteration of labeling conditions affected conjugation efficiencies. By

labeling FhuD T181C overnight with AEDANS at 4 ˚C, two moles of label

incorporated for every mole of protein. FhuD contains a single endogenous Cys-

237; based upon the X-ray crystal structure, this residue is not expected to be

solvent-exposed. To determine if Cys-237 were reactive to conjugation, we

labeled FhuD using the conditions above; unexpectedly, FhuD was labeled. Cys-

237 localizes outside the phage display-identified TonB-binding surfaces. Given

the reactivity of Cys-237 toward conjugation, we used this conjugate to exclude

the possibility that TonB binds to regions other than those implicated by phage

display. Addition of TonB to FhuD-AEDANS caused an insignificant increase in

the fluorescence emission of AEDANS (data not shown). Given this outcome that

contrasts with FhuD T181C-AEDANS, we consider that Cys-237 is not part of a

TonB-binding environment. Our conclusion is that TonB-binding localizes to

regions on FhuD that were identified by phage display.

3.3.5 Detection of a TonB–FhuD complex by surface plasmon resonance

Our previous use of SPR technology quantified binding of FhuA to

immobilized TonB (77). Given the outcomes of this experimental design, we

adapted its use for the study of TonB–FhuD interactions. Dose-dependent

binding of FhuD to amine- and thiol-coupled TonB surfaces was observed

Page 157: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

133

(Figure 3.7). Purified FhuA (0.1-1 M) also bound to immobilized TonB as a

positive control; FhuD binding (0.5 M) was unaltered in the presence of BSA

(0.5 M) as a competitor (data not shown). To improve kinetic analyses, a

homogeneous presentation of a lower density thiol-coupled Cys-TonB surface

was utilized. A high-affinity interaction (KD ~20 nM) between FhuD and TonB

was determined by SPR using a single binding site model. This interaction was

characterized by slow association (ka, ~2 x 104 M

-1s

-1) and slow dissociation (kd,

~4 x 10-4

s-1

) rates. Presence of the siderophore Fcn did not significantly alter the

affinity between FhuD and TonB by SPR (Figure 3.7B).

For reasons analogous to those considered with fluorescence data, we

attempted to fit our SPR data to a model describing multiple binding sites. SPR

data were fit to the heterogeneous ligand model, a model previously used to

distinguish independent binding sites on biological macromolecules (185,186).

Similar to fluorescence, fits of the data to this model quantified two binding sites;

a high affinity site with KD in the low nanomolar range and a weak affinity site

with KD in the low micromolar range. However, uncertainties associated with

these fits confounded their interpretation. Because of this outcome we favour a

single site of interaction between TonB and FhuD.

By having established formation of a TonB–FhuD complex, we performed

multi-component SPR analyses to identify a ternary FhuA–TonB–FhuD complex.

Initially, either a TonB–FhuA complex or a TonB–FhuD complex was formed by

injecting each analyte over amine-coupled TonB surfaces. Once these binary

complexes had formed, the secondary analyte was then injected.

Page 158: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

134

Figure 3.7. Real-time kinetics of TonB–FhuD binding interaction detected by SPR. A.

Representative SPR sensogram for FhuD (top to bottom: 1000, 500, 250 and 100 nM) binding to

amine-coupled TonB (250 RU) in the absence of Fcn. B. Representative SPR sensogram for FhuD

(top to bottom: 1000, 500, 250 and 100 nM) binding to thiol-coupled Cys-TonB (48 RU) in the

absence (blue) or presence (red) of a tenfold molar excess of Fcn.

Injection of FhuD over a preformed TonB–FhuA complex indicated formation of

a ternary FhuA–TonB–FhuD complex (Figure 3.8A). Similarly, injection of

FhuA over a previously formed TonB–FhuD complex indicated formation of a

ternary FhuA–TonB–FhuD complex (Figure 3.8B). Ternary complexes formed

independent of the order of analyte addition. Qualitatively, association and

dissociation rates were also independent of the order of analyte addition and

mirrored those rates observed upon formation of each respective binary complex.

Page 159: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

135

Figure 3.8. Multicomponent SPR analysis to detect ternary complex formation between FhuA–

TonB–FhuD. A. SPR sensogram indicating (I) baseline for buffer flowing over amine-coupled

TonB (250 RU); (II) increased signal change due to binding of FhuA (1 M); (III) stable FhuA–

TonB complex after a 0.5 M NaCl wash; (IV) increased signal change due to binding of FhuD (1

M); (V) return to baseline after regeneration. B. SPR sensogram indicating (I) baseline for

buffer flowing over amine-coupled TonB (250 RU); (II) increased signal change due to binding of

FhuD (1 M); (III) increased signal change due to binding of FhuA (1 M); (IV) return to baseline

after regeneration.

3.4 Discussion

Translocation of siderophores into the cytoplasm of Gram-negative

bacteria is partially understood, mainly with respect to the interplay between

TonB and TonB-dependent OM receptors. However, molecular events occurring

after translocation of siderophore into the periplasm remain largely unknown.

Page 160: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

136

Binding of the translocated iron-bound siderophore to the periplasmic binding

protein may be determined purely by diffusion within the periplasm. However,

this mechanism poorly accounts for the weak affinity exhibited by FhuD toward

its ligands. Sprencel et al. determined (187) a value of approximately 4,000

copies of the ferric enterobactin periplasmic binding protein FepB in

E. coli, a low value compared to other periplasmic binding proteins such as those

involved in sugar or amino acid transport. Köster and Braun (174) demonstrated

that chromosomally-encoded FhuD was undetectable from silver-stained SDS-

PAGE gels of periplasmic extracts. Taken together with the modest KD of 1.0 M

(113), these data suggest that diffusion alone would be insufficient to account for

unidirectional siderophore transport. In addition to siderophore capture,

diffusion-governed docking of siderophore-bound FhuD to the CM permease may

be an inefficient process.

This study highlights novel interactions in the periplasm that are involved

in siderophore uptake by E. coli. By exploiting phage display and adopting three

biophysical strategies, we identified and mapped an interface between two

interacting protein partners. On FhuD, a TonB-binding surface was identified that

partially overlaps the FhuD siderophore binding site. On TonB, three distinct

regions of FhuD-binding surfaces were identified, two of which localized to

regions for which no structural data exists. Given apparent concordance between

phage display(180), and structural biology (96), the regions identified in this study

imply specific interactions between TonB and FhuD.

Page 161: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

137

Interactions between TonB and FhuD are multidimensional. By DLS, we

identified a 1:1 TonB–FhuD complex. Fluorescence spectroscopy indicated an

apparent affinity for this complex to lie within the mid-nanomolar range. This

affinity contrasts with the low nanomolar range determined by SPR. Our current

SPR studies for FhuD–TonB are similar to the previous experimental design used

to monitor the TonB–FhuA kinetics in real-time (77). The affinity range

predicted for TonB–FhuD interactions by SPR (low nM) is consistent with the

previously reported range (94) of TonB–FhuA interactions also by SPR. The

differences in reported affinities between fluorescence and SPR may be attributed

to solution-phase versus immobilized systems or to buffer requirements of the

different technologies. Given these differences, our data indicates that the affinity

between TonB and FhuD lies somewhere in the low to mid-nanomolar range.

Significantly, the presence of Fcn did not alter the affinity between TonB

and FhuD; there was no evidence for competition. However, the TonB-binding

surface on FhuD as identified by phage display both overlaps the siderophore

binding site and extends beyond it; competition would not be expected.

Furthermore, there is no evidence to suggest large conformational changes in

FhuD upon binding siderophore (119). In the siderophore-bound state, FhuD

probably maintains a rigid backbone; this rigidity would not influence binding of

TonB. One implication of these findings is that interactions between siderophore

and FhuD are distinct from interactions between TonB and FhuD. It remains to

be determined whether siderophore can still bind to FhuD when complexed with

Page 162: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

138

TonB, or whether bound TonB prevents siderophore binding through occlusion of

FhuD’s siderophore binding site.

A regulated mechanism of siderophore transport would involve

coordination of protein–protein interactions that would facilitate direct transfer of

siderophore among protein partners. For such a mechanism, siderophore transfer

would follow a sequence of directed exchanges from OM receptor, to periplasmic

binding protein, to CM permease. Directed transfer of this sort would require a

scaffold whereby protein–protein interactions drive spatial and temporal

localization of the periplasmic binding protein to regions involved in siderophore

translocation activities. The dynamic nature by which TonB cycles or changes its

conformation during energy transduction offers itself as an ideal candidate to

fulfill this role.

Our SPR data indicated formation of a ternary FhuA–TonB–FhuD

complex. This finding, taken together with the recent structural determinations of

TonB bound to FhuA (96) and to BtuB (97) underscores the possibility of such a

complex. It would position a periplasmic binding protein to accept siderophore

immediately after its translocation through a TonB-dependent OM receptor.

Examination of the TonB–FhuA interaction surface provides a means to evaluate

residues involved in the protein–protein interaction. In the TonB–FhuA crystal

structure, residues N-terminal to Arg-158 on TonB were not resolved and may not

participate in the TonB–FhuA interface. The FhuD-interacting residues on TonB

that were predicted by phage display (region III, Figure 3.3C) localize

Page 163: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

139

immediately N-terminal to TonB Arg-158. We infer that these residues are poised

to bind FhuD in a FhuA–TonB–FhuD ternary complex.

Given these experimental outcomes, we propose a structural model for a

FhuA–TonB–FhuD complex. Examination of complementary binding regions on

solvent-accessible surfaces for known TonB and FhuD structures reveals no

obvious means by which the two proteins can interact. However, one can

rationally position FhuD to the TonB–FhuA structure using these surfaces as

docking constraints. Figure 3.9 depicts a model for a ternary FhuA–TonB–FhuD

complex that is based on our experimental evidence. In this model, FhuD-binding

regions on TonB are coloured according to the convention adopted in Figure 3.4.

TonB residues 153

PRALS157

corresponding to region III (Figure 3.3C) were

computationally modeled at its N-terminus. Their position illustrates that

interaction with FhuD at this region would result in the apposition of its

siderophore binding site with the FhuA lumen such that it would intersect with the

trajectory of the translocated siderophore. Furthermore, TonB residues interacting

with FhuA in the TonB–FhuA crystal structure are distal to those TonB residues

which contact FhuD. Separation of these binding surfaces may therefore

coordinate transduction of energy to FhuA with directed localization of FhuD

beneath the FhuA lumen.

The precise sequence of these events has yet to be established is. Our data

cannot distinguish whether FhuD remains bound to TonB during the energy

transduction cycle or if binding is a transient event. One possibility is that FhuD

binds and dissociates as a function of the TonB energy transduction cycle,

Page 164: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

140

Figure 3.9. Model of a FhuA-TonB-FhuD ternary complex. Stereo image depicting a possible

ternary complex between FhuA, TonB and FhuD. FhuD (PDB code 1EFD) was manually docked

under the TonB-FhuA crystal structure (PDB code 2GRX) using phage display-identified protein–

protein interaction surfaces as docking constraints. Complementary phage display-identified

surfaces are coloured blue on both TonB (yellow, surface representation) and FhuD (salmon,

ribbon representation). The orientation localizes the FhuD siderophore binding site beneath the

lumen of FhuA (green, ribbon representation). For clarity, a molecular surface is projected on

TonB.

perhaps resulting from the β-strand exchange that TonB is known to undergo

(72,74,75,96,97). Prolonged association of FhuD with TonB seems unlikely as

FhuD must ultimately deliver siderophore to FhuB/C. It is intriguing that the

most extreme N-terminal FhuD-binding surface on TonB is at a region close to

probable contact sites with ExbB/ExbD. Such placement may be a means by

which TonB directs transfer of FhuD from the OM to CM as TonB disengages

Page 165: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

141

FhuA. This mechanism would ensure that FhuD samples a space proximal to the

CM, thereby enhancing the probability of encountering FhuB.

Previous reports have indicated (114,188) that interactions between the

FhuD homolog BtuF and its cognate transporter BtuC/D are virtually irreversible.

This observation remains to be clarified in light of our experimental evidence,

which for the first time identifies TonB as a binding partner for periplasmic

binding proteins. We advocate a mechanism favouring transient associations of

periplasmic siderophore-binding proteins with TonB and with their cognate CM

transporters at discrete steps during the siderophore transport cycle.

We previously used phage display in concert with biophysical methods to

map unambiguously the network of protein–protein interactions that occur at the

TonB–FhuA interface. These outcomes were recently confirmed by the X-ray

structure of the TonB–FhuA complex. This strategy is now extended to identify

interfaces involved in TonB–FhuD interactions. Such interactions may serve to

coordinate spatial and temporal localization of periplasmic binding proteins to

environments involved in siderophore uptake. Given the diversity of components

among different siderophore transport systems, we propose that in addition to its

role as energy transducer, TonB acts as a unifying element, a scaffold to regulate

the unidirectional flow of iron-bound siderophore from the OM to the CM.

3.5 Acknowledgements

Research was supported by operating grant MOP-62774 to J.W.C. from

the Canadian Institutes of Health Research (CIHR). D.M.C. was recipient of the

Page 166: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

142

McGill Graduate Studies Fellowship and an F.C. Harrison Fellowship,

Department of Microbiology and Immunology. Natural Sciences and Engineering

Research Council (Canada) provided an Undergraduate Student Research Award

to I.R.M. and a Post Graduate Scholarship to J.-N.G. É.M. was trainee from the

École supérieure de biotechnologie Strasbourg. A.C. won a Traveling Award for

Research Training from the National Health and Medical Research Council

(Australia). Canada Foundation for Innovation awarded infrastructure to the

Montreal Integrated Genomics Group for Research on Infectious Pathogens.

Sheldon Biotechnology Centre at McGill University is supported by multi-user

maintenance grants from CIHR. We appreciate contributions of experimental

materials by M. Damlaj, J. Deme, J. Gilbert, K. James and C. Ng; and editorial

support by J.A. Kashul.

Page 167: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

143

Preface to chapter 4

In chapter 3, we demonstrated that TonB and FhuD form a complex.

Regions within TonB that are essential for this interaction are examined in chapter

4. We characterized the properties of periplasmic TonB derivatives and asked

how these properties affect binding to FhuD. By SPR, we determined that neither

TonB’s N-terminal region nor its proline-rich region is required for interaction

with FhuD. These findings suggested that TonB’s central and C-terminal regions

were essential for interaction with FhuD. We then computationally modeled how

such regions might bind to FhuD. Our model suggested that when bound to

TonB, FhuD’s siderophore binding site could project towards the OM, where its

orientation could facilitate siderophore capture as it emerges during transport.

Page 168: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

144

Chapter 4

C-terminal region of TonB positions periplasmic binding protein

FhuD for siderophore transport in Escherichia coli

David M. Carter, Justin C. Deme, Mark A. Hancock and James W. Coulton

Manuscript submitted July, 2009

Page 169: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

145

4.0 Summary

The Ferric hydroxamate uptake (Fhu) system of Escherichia coli actively

transports ferric hydroxamate-type siderophores. The main components of the

Fhu system include outer membrane receptor FhuA, periplasmic binding protein

FhuD, cytoplasmic membrane-embedded permease FhuB/C, and the energy

transducing TonB–ExbB–ExbD complex. Recently, we demonstrated that TonB

binds FhuD. Here we extend these analyses by delineating regions within TonB

that are essential for this interaction. By analytical ultracentrifugation and

fluorescence spectroscopy, we characterized properties of three periplasmic TonB

variants: a derivative possessing residues 33–239; a derivative with a deletion

(residues 66–100) of the central proline-rich region; and a truncated derivative

possessing residues 103–239. Our analyses indicate that all derivatives are

elongated monomers, consistent with knowledge that TonB possesses a structured

C-terminal domain and unstructured central and N-terminal regions. By surface

plasmon resonance, all TonB derivatives exhibit similar low nanomolar binding

affinities for FhuD. Hence, essential FhuD-binding determinants localize within

predominantly unstructured regions of TonB. To further characterize the nature

of this interaction, we computationally docked onto the surface of FhuD, an

oligopeptide that corresponds to an unstructured region of TonB and that was

predicted to bind FhuD. These simulations place the peptide against FhuD at

predicted TonB-binding surfaces. Extension of N- and C-termini of the lowest

energy docked peptide indicates that when bound to TonB, FhuD’s siderophore

binding site orients towards the outer membrane.

Page 170: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

146

4.1 Introduction

Most bacteria require the essential nutrient iron in order to grow and

divide. However, the bioavailable concentration of iron is bacteriostatic due to its

propensity to form insoluble ferric hydroxides in the environment and due to

sequestration of labile iron pools by host iron-binding proteins (6,7). Given these

limitations, bacteria have evolved high affinity iron uptake systems whereby they

secrete siderophores capable of scavenging iron under iron-deplete conditions.

Having bound iron with high affinity, ferric-siderophores are transported back

into a bacterium by various families of siderophore transport systems (145). The

ferric hydroxamate uptake (Fhu) system of Escherichia coli is responsible for

uptake of hydroxamate siderophores including ferricrocin. The main components

include outer membrane (OM) siderophore receptor FhuA, periplasmic binding

protein (PBP) FhuD, and cytoplasmic membrane (CM)-embedded ATP permease

FhuB/C.

The structure of FhuA (42) revealed a two-domain protein comprising an

N-terminal cork domain that independently folds and inserts into a C-terminal β-

barrel domain. Passive transport of siderophore through FhuA is prevented by

occlusion of its lumen with the cork domain. Transport requires energy-

dependent rearrangement of FhuA’s cork domain so that a pore large enough to

allow translocation can form. The CM-associated TonB–ExbB–ExbD multi-

protein complex provides the energy input required to facilitate this structural

rearrangement. By harnessing the proton motive force, the TonB–ExbB–ExbD

Page 171: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

147

complex conformationally activates TonB in a manner that promotes energy

transduction to siderophore-bound FhuA.

Structural information is incomplete for the TonB–ExbB–ExbD complex.

TonB is a modular 239-residue protein embedded within the CM by a 33-residue,

N-terminal transmembrane helix. It interacts with ExbB and ExbD through this

single helix (85) in a TonB:ExbB:ExbD stoichiometry postulated to be 1:7:2. (82)

The remainder of TonB is periplasmic and mostly unstructured. Central to the

periplasmic domain is a proline-rich region consisting of Lys-Pro, Glu-Pro

repeats, considered to adopt an extended structure based on an NMR study of the

isolated residues (70). This may serve to span the periplasm; however, deletion of

this region only marginally reduced TonB-dependent transport (71). The C-

terminal 89 residues of TonB form a structured domain responsible for energy-

independent interactions with TonB-dependent OM receptors (125).

Structural studies demonstrated that TonB’s C-terminal domain is

dynamic. The first crystal structure of a TonB derivative, residues 155–239,

revealed a tightly intertwined dimer (72). Analytical ultracentrifugation (AUC)

studies (73,77) and a second crystallographic study

(73) later confirmed this

finding. A slightly longer TonB derivative possessing residues 148–239 also

crystallized as a dimer (74), albeit of loosely associated monomers.

Unexpectedly, this study also demonstrated that the same derivative was

monomeric by AUC. A longer derivative of TonB possessing residues 103–239

was demonstrated to be monomeric by NMR spectroscopy (75). Most recently,

another structure of a monomeric TonB variant from the fish pathogen Vibrio

Page 172: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

148

anguillarum was solved by NMR (76). From these studies it is apparent that

derivatives of TonB possessing at least residues 148–239 prevent dimerization in

solution, probably by altering distal conformations of a C-terminal hinge region

centered on residues 231

KING234

of E. coli TonB.

In addition to binding TonB-dependent receptors, TonB also binds the

PBP FhuD (189) and probably binds other PBPs involved in TonB-dependent

transport. By phage display technology, we identified complementary regions

between TonB and FhuD that were predicted to interact and we then demonstrated

complex formation in vitro. Here we extend these results by delineating regions

within TonB that are essential to forming a complex with FhuD. Through a series

of periplasmic TonB deletions, we investigated the importance of each region

predicted to bind FhuD. First, structural properties of each TonB derivative were

investigated to ascertain whether the length of TonB influenced its overall shape

and tertiary structure and whether these differences could affect binding of FhuD.

Second, SPR was used to examine the affinity between each TonB derivative and

FhuD. Finally, a computational model was generated to predict the way in which

a given TonB-derived, FhuD-binding peptide might bind to FhuD. Our results

indicate that unstructured regions on TonB, proximal to the structured C-terminal

domain, constitute the minimal elements required to bind FhuD and further

suggest that these regions bind FhuD in a manner that orients FhuD’s siderophore

binding site towards the OM.

Page 173: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

149

4.2 Materials and methods

4.2.1 Bacterial strains and plasmids

E. coli strains BL21 DE3 (pLysS) and ER2566 were used for protein

expression. Plasmids pWA01 (77), pWA02

(94) and pDMC01 (this study) were

used to express TonBs 33–239, Δ66–100 and 103–239 respectively. Plasmid

pCMK02 (94) was used to express a TonB Δ66–100 variant possessing an N-

terminal cysteine for SPR analysis. Plasmid pMR21, a gift from W Köster

(VIDO, SK), was used to express a decahistidine-tagged version of FhuD.

Plasmid pMal-pIII was used to express maltose binding protein.

4.2.2 Cloning of TonB 103–239

A periplasmic region of TonB corresponding to residues 103–239 was

sub-cloned from pWA01, which contains residues 33–239. Primers

corresponding to the appropriate region were synthesized (Alpha DNA, Montreal)

and the region PCR-amplified. The PCR product was ligated into digested pJD01

(unpublished data, a mutated version of pMR21 with a TEV protease-cleavable

His-tag) to generate pDMC01, a decahistidine-tagged product of TonB 103–239

plus a TEV protease cleavage site.

4.2.3 Protein expression

Bacterial cultures harbouring expression plasmids were grown at 37°C

overnight in LB broth (Fisher Scientific) supplemented with appropriate

antibiotics. Cultures were then diluted 100-fold into 6 × 1 l each of fresh LB

Page 174: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

150

broth plus antibiotics and grown at 37°C until mid-log phase. Protein expression

was induced by addition of IPTG (BioVectra, Charlottetown, PE) to final

concentrations of either 0.5 mM (TonB 33–239 and Δ66–100), 0.4 mM (TonB

103–239) or 1 mM (FhuD). Upon induction, cells were grown for either four

hours at 30°C (TonB 33–239 and Δ66–100), one hour at 37°C (TonB 103–239) or

overnight at 37°C (FhuD). Cells were then harvested by centrifugation of 3 l each

from the 6 l culture and the resulting pellets were stored at -20°C until further use.

Maltose binding protein was expressed as previously described (180).

4.2.4 Protein purifications

TonBs were purified by first thawing a frozen cell pellet in 40 ml of buffer

Hni (50 mM Hepes pH 7.5, 30 mM NaCl and 5 mM imidazole (Fluka)). Once

thawed, the cell paste was supplemented with RNAse, DNAse, MgCl2, lysozyme,

PMSF, and a Complete EDTA-free protease inhibitor tablet (Roche). Cells were

then lysed by two passages through an Emulsiflex (Avestin) and clarified by

centrifugation at 27,000 × g for 1 h at 4°C. The supernatant was further clarified

by passage through a 0.45-µm filter and loaded onto a column containing 25 ml of

Ni-NTA superflow (Qiagen) equilibrated in buffer Hni. After a 500 mM salt

wash, contaminants were removed with a step to 50 mM imidazole (Fluka).

TonBs were then eluted with a linear gradient up to 500 mM imidazole over four

column volumes (CV). The Ni-NTA eluate was pooled, supplemented with 3

mM EDTA plus an additional 175 µM PMSF and applied to a 5 ml Source S

strong cation exchange column (GE Health Sciences) equilibrated in buffer Hn

Page 175: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

151

(50 mM Hepes pH 7.5 plus 30 mM NaCl). After a wash, TonBs were eluted with

a gradient up to 200 mM NaCl over 10 CV followed by a 1 CV gradient up to 500

mM NaCl and an additional 1 CV gradient up to 1 M NaCl. FhuD was purified as

described previously (189) and maltose binding protein was purified as described

previously (180).

4.2.5 Analytical ultracentrifugation

Prior to AUC data collection, TonBs were dialyzed overnight against a

buffer containing 50 mM Tris pH 7.5 and 150 mM NaCl. Sedimentation velocity

experiments were conducted on a Beckman XL-I analytical ultracentrifuge (77).

Data were fit to a c(s) model using the program Sedfit (143).

4.2.6 Fluorescence spectroscopy

Prior to fluorescence data collection, TonBs were dialyzed overnight

against the same buffer used in AUC experiments. TonBs were then diluted to

3.5 µM in dialysis buffer and centrifuged for 10 min at 4°C. Spectra were

recorded on a Varian Cary Eclipse fluorimeter with the following settings: λex =

280 nm, excitation slit = 2.5 nm, λem = 300–400 nm, emission slit = 5 nm,

averaging time = 0.1 sec, scan rate = 50 nm/min and a detector voltage of 700 V.

A total of 10 scans were collected at 25°C and averaged. Spectra were corrected

by subtraction of background fluorescence due to buffer alone. Protein

concentrations were normalized by a Bradford assay (Bio-Rad) and by

measurement of absorbance at 280 nm on a Varian Cary Bio 1 spectrophotometer.

Page 176: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

152

4.2.7 Surface plasmon resonance

Binding interactions between FhuD (~32 kDa) and TonB (~25 kDa TonB

33–239; ~21 kDa TonB Δ66–100; ~18 kDa TonB 103–239) were examined using

label-free, real-time Biacore 3000 instrumentation (GE Healthcare Bio-Sciences

AB). Prior to all SPR experiments, purified protein preparations were dialyzed

against 50 mM Hepes pH 7.4 containing 150 mM NaCl. Experiments were

performed on research-grade CM4 sensor chips at 25°C using filtered (0.2 µm)

and degassed HBS-ET running buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 3

mM EDTA, 0.05% (v/v) Tween 20). Protein-grade detergents (Tween 20,

TritonX-100, Empigen) were from Anatrace; all other chemicals were reagent-

grade quality. As previously described (189), TonB derivatives (3 μg/ml in 10

mM sodium acetate pH 5.5) and corresponding reference surfaces were

immobilized using Biacore Amine or Thiol Coupling Kits as recommended by the

manufacturer. To assess binding specificity and kinetics, PBPs (0–500 nM FhuD

and negative MBP control) were titrated over immobilized surfaces at 30 μl/min

using 'KINJECT' mode (1 min association +/- 15 min dissociation). For binding

assays performed in the presence of siderophore, a 10-fold molar excess of

ferricrocin was added to FhuD and incubated for at least 30 min prior to injection.

In all cases, surfaces were regenerated between titration series at 50 μl/min using

two 30 s pulses of solution I (HBS-ET containing 0.5 M NaCl, 5 mM NaOH,

0.05% (v/v) Triton-X100) and solution II (HBS-ET containing 0.5 M NaCl, 5 mM

NaOH, 0.05% (v/v) Empigen), followed by 'EXTRACLEAN' and 'RINSE'

procedures. SPR data are representative of duplicate injections acquired from

Page 177: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

153

three independent trials. Mass transport-independent data were double-referenced

(183) and analyzed according to a “1:1 Titration” model in the BIAevaluation

v4.1 software (190).

4.2.8 Computational docking

Oligopeptides corresponding to TonB residues 123

SPFENTAPARL133

(predicted FhuD-binding region II) and 153

PRALSRNQ159

(predicted FhuD-

binding region III) were used as flexible ligands and were docked to the crystal

structure of FhuD (PDB code 1EFD) (111) with the program AutoDock 4 (191).

The TonB region II peptide was modeled as an α-helix with the program Pymol

v1.1r1 (170). Two glycines were attached to each terminus to act as inert spacers

that should not influence binding specificity during simulation. Prior to docking,

the α-helical peptide was minimized using an AMBER force field implemented in

the program UCSF Chimera v1.3 (192). For docking, the peptide was given 12

torsional degrees of freedom along selected sidechains. Additionally, the

backbone was allowed some torsional freedom to relax during the course of

simulation. The TonB region III ligand was derived from crystal structure PDB

code 1U07 (74). Residues comprising region III were truncated from the PDB

coordinates, capped with glycines and minimized as with the region II peptide.

Selected sidechains and backbone atoms were also given torsional freedom to

relax during the course of simulation. Two separate simulations consisting of 50

runs each were performed. A Lamarckian genetic algorithm (171) was used in

each run to dock the peptides; 25,000,000 energy evaluations were performed

Page 178: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

154

with a population size of 300 over 29,000 generations, a mutation rate of 0.02,

and a crossover rate of 0.8.

4.3 Results

4.3.1 TonB derivatives are elongated monomers with similar elements of tertiary

structure

Previously, we demonstrated that a periplasmic derivative of TonB

interacts with FhuD to form a 1:1 siderophore-independent complex (189). To

extend these results, we investigated whether truncations of TonB could affect its

FhuD-binding properties. Three periplasmic TonB variants were characterized

(Figure 4.1): a derivative possessing residues 33–239 (denoted hereafter as TonB

33–239); a derivative with an internal deletion of TonB’s proline-rich region

(TonB Δ66–100); and a third derivative possessing residues 103–239 (TonB 103–

239). Both TonB 33–239 and TonB Δ66–100 retained the three predicted FhuD-

binding regions (regions I–III respectively, Figure 4.1B and C), whereas TonB

103–239 retained the second and third FhuD-binding regions, respectively

(regions II and III, Figure 4.1D). All derivatives were isolated to near

homogeneity (at least 95% purity) as assessed by SDS-PAGE. Similarly, FhuD

was isolated to near homogeneity.

To understand the influence of hydrodynamic shape on formation of a

TonB–FhuD complex, we characterized the frictional ratios of the various TonB

derivatives by sedimentation velocity. Our earlier studies (77,94) determined the

frictional ratios of TonB 33–239 and TonB Δ66–100. Both values (f/fo = 2.39 for

Page 179: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

155

TonB 33–239; f/fo = 2.27 for TonB Δ66–100) indicated that these TonB

derivatives are elongated, consistent with knowledge that TonB must span the

periplasm for energy transduction. In this study, we similarly modeled frictional

ratios of each derivative using a continuous distribution c(s) model in Sedfit

(143). In agreement with our previous analyses, TonB 33–239 is elongated and

possesses a frictional ratio of 2.36; RMSD = 0.0041. Similarly, TonB Δ66–100 is

also elongated and possesses a frictional ratio of 2.02; RMSD 0.0046. The

shortest TonB 103–239 exhibits a smaller frictional ratio of 1.80; RMSD =

0.0044, indicating that it is considerably less elongated than the other derivatives,

but still deviates from values typically associated with globular proteins (f/fo

~1.3).

Figure 4.1. Schematic representations of TonB derivatives from this study. A. Full-length TonB.

TM: transmembrane domain, PRR: proline-rich region. Predicted FhuD-binding regions I–III are

indicated in Roman numerals. TonB amino acid numbering is indicated above the schematic. N-

and C-termini are labeled. B. TonB 33–239. Schematic is labeled according to panel A. N-

terminal histidine tag is indicated. C. TonB Δ66–100. Schematic is labeled as in panel B. D.

TonB 103–239. Schematic is labeled as in panel B.

Page 180: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

156

Stoichiometries of TonB derivatives were also determined. Our previous

analyses demonstrated that both TonB 33–239 and TonB Δ66–100 sediment as

monomers in solution. Our current analyses agree with these previous reports and

indicate that both TonB 33–239 and TonB Δ66–100 are monomeric in solution.

Consistent with the NMR structure (75) of a similar derivative, TonB 103–239 is

also monomeric in solution. Our data indicate a hydrated molecular weight of

19.0 kDa, in agreement with the theoretical molecular weight (18.4 kDa) of

decahistidine-tagged TonB 103–239 monomer.

To determine whether the derivatives possessed similar elements of

tertiary structure, we used intrinsic fluorescence spectroscopy to examine the

environments surrounding fluorophores in each TonB derivative. Of the ten

aromatic residues within TonB’s primary sequence, six localize within the

structured C-terminal domain. If each derivative were to adopt a similar fold,

fluorescence spectra should be identical. When prepared at equimolar

concentrations, all TonB derivatives possess similar spectra, with overlapping

maxima centered at 335 nm (data not shown). This indicates that all TonB

derivatives possess similar folds within their C-termini. Together with AUC data,

which demonstrates that all TonB derivatives are elongated monomers, we

interpret our fluorescence results to mean that all periplasmic derivatives of

monomeric TonB have unstructured N-terminal regions and a structured C-

terminal region.

Page 181: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

157

4.3.2 TonB derivatives bind FhuD with equal affinities

Using the single-cycle kinetics approach (190), binding interactions

between immobilized TonB derivatives and FhuD were examined with label-free,

real-time SPR. FhuD exhibits specific, concentration-dependent binding to all

TonB surfaces (Figure 4.2). As a negative control, there was little or no non-

specific binding to any of the surfaces when another PBP, maltose binding protein

(MBP), was titrated under identical assay conditions. FhuD binding to all

derivatives is unaltered in the presence of siderophore (data not shown) and, when

analyzed according to a “1:1 Titration” model in the BIAevaluation software,

similar kinetic (slow on- and slow off-rates) and affinity (low nanomolar)

constants are observed (Table 4.1). Accounting for differences in the molecular

weight, coupling chemistry, and surface density of each derivative, the observed

binding responses (RU and subsequent affinity calculations) indicate that all three

TonB derivatives bind FhuD in a similar manner.

Figure 4.2. Single cycle kinetic analysis of FhuD binding to TonB derivatives using label-free,

real-time SPR. FhuD was titrated (0–500 nM; 30 µl/min × 1 min association ± 15 min

dissociation) over immobilized surfaces: red, 180 RU amine-coupled TonB 33–239; cyan, 100 RU

thiol-coupled TonB Δ66–100; yellow, 150 RU amine-coupled TonB 103–239. For all TonB

derivatives, MBP (green, 0–500 nM) exhibited little or no non-specific binding. Coloured lines

represent experimental data and black lines represent best fit to “1:1 titration” model.

Page 182: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

158

Table 4.1. Kinetics and affinity of TonB–FhuD interactions according to “1:1 titration”

model

†Reported errors for apparent rate constants represent the standard deviation from three

independent SPR trials.

4.3.3 Computational models predict the orientation of FhuD when bound to TonB

For the predicted FhuD-binding regions on TonB, our SPR results

demonstrated that region I was not essential to form a TonB–FhuD complex.

Since the smallest TonB regions that bind to FhuD were likely to be regions II and

III, we computationally modeled how the peptides might bind to FhuD. Two

approaches were employed to test this hypothesis. First, we took advantage of

currently available TonB structural models; regions N-terminal to residue 150 are

considerably flexible and unstructured. Given this knowledge, we modeled the

core of TonB region II, consisting of residues 123

SPFENTAPARL133

, as an α-

helical oligopeptide. After energy minimization, the TonB oligopeptide was

docked to the crystal structure of FhuD using PDB code 1EFD (111) and the

program AutoDock 4 (191). During simulation, the peptide was given torsional

degrees of freedom and allowed to sample space that was limited to the side of

FhuD that our phage display analysis predicted it would bind.

Two separate simulations gave low energy solutions centering the TonB

peptide on the predicted FhuD-binding surface (Figure 4.3A). Results focus on

the two lowest energy solutions observed in each of the two simulations. Both

solutions predict that the peptide will spontaneously bind, with energies ranging

TonB derivative ka × 103 (M

-1 s

-1)† kd × 10

-5 (s

-1)† KD × 10

-9 (M)

TonB 33–239 7.1 ± 1.2 5.0 ± 3.1 7.0 ± 1.8

TonB Δ66–100 3.5 ± 0.3 2.6 ± 1.2 7.5 ± 1.1

TonB 103–239 4.2 ± 0.8 5.4 ± 1.7 13 ± 5.9

Page 183: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

159

from -2.39 kcal/mol to -1.06 kcal/mol and -2.53 kcal/mol to -1.35 kcal/mol for the

first and second simulation respectively. These values are consistent with

magnitudes of energies involved in salt bridge formation or in hydrogen bond

formation.

While orientations vary for low energy docked peptides, N-termini from

the four lowest energy region II conformers center within 7 Å of FhuD residue

Leu-50. This falls within a cavity on FhuD that exhibits elements of net

electronegativity as well as nonpolarity (Figure 4.3B). The cavity is also

proximal to the first FhuD region that our previous phage display analysis

predicted would serve as a TonB-binding surface. Significantly, lowest energy

TonB region II peptides orient so as to promote biologically constructive

interactions with FhuD. TonB is anchored to the CM by its N-terminal

transmembrane domain, while its C-terminus projects toward the OM. By

extending the peptide backbone trajectory of the C-termini of lowest energy

docked region II peptides, TonB’s C-terminus could project towards the vicinity

of FhuD’s siderophore binding site. Similarly, extension of the N-termini of

lowest energy docked peptides could project TonB’s N-terminus towards the CM.

These results support a model where upon binding to TonB, FhuD’s siderophore

binding site orients towards the OM.

The second docking approach used structural data corresponding to the

most complete structure of TonB (PDB code 1UO7) (74). Three-dimensional

coordinates that correspond to TonB region III (residues 153

PRALSRNQ159

) were

Page 184: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

160

Figure 4.3. TonB region II peptide docked to the surface of FhuD. A. Lowest energy TonB-

derived oligopeptide (binding energy -2.53 kcal/mol) bound to the surface of FhuD (blue). The

phage display-predicted TonB-binding surface is coloured red. The peptide N-terminus is marked

with a white arrow; B. electrostatic surface representation of FhuD, contoured from -2 kT (red) to

+2 kT (blue). The predicted TonB-binding surface is outlined in black; C. temperature factor

distribution of FhuD. Temperature factors derived from FhuD PDB file 1EFD are mapped onto

the surface of FhuD. Colours are contoured such that dark blue represents regions of low

uncertainty (B-factors ~13), green represents regions of intermediate uncertainty (B-factors ~23)

and red represents regions of greatest uncertainty (B-factors ~41). The predicted TonB-binding

surface is outlined in black.

Page 185: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

161

excised from the crystal structure model and used as a ligand that we then docked

to FhuD. However, docking solutions with this peptide produced only positive

binding energies (data not shown).

4.4 Discussion

Our previous phage display and biophysical analyses demonstrated that, in

addition to binding TonB-dependent OM receptors such as FhuA, TonB also

binds PBPs such as FhuD. Using three periplasmic derivatives, we now identify

TonB regions that are important for binding FhuD. Our hydrodynamic analyses

of the TonB derivatives indicate that all are monomeric and elongated in solution.

These data are consistent with the NMR structure of TonB 103–239, which

revealed a monomer with a structured C-terminal domain plus an unstructured and

presumably extended N-terminal region. A shorter TonB derivative possessing

residues 148–239 was also monomeric in solution (73), yet crystallized as a dimer.

In contrast, shorter derivatives with residues 155–239 and fewer dimerize

in solution (77). Given their propensity to form dimers, we did not attempt to

characterize binding of FhuD to shorter TonB derivatives. We reasoned that

removal of predicted FhuD-binding regions II and III could promote TonB

dimerization. If such a derivative did not bind FhuD, it might be due to

dimerization and not due to loss of predicted FhuD-binding regions. Furthermore,

we did not attempt to model the hydrodynamic properties of TonB–FhuD

complexes. Our c(s) analyses measured global weight-averaged frictional ratios,

Page 186: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

162

which would necessitate further deconvolution in order to gain insight into the

frictional ratio of the TonB–FhuD complex.

Our fluorescence results indicate that tertiary structural features are

superimposable amongst the derivatives. These elements probably correspond to

the structured region observed in the TonB 103–239 NMR structure.

Accordingly, the spectra of derivatives examined here probably arise due to

positioning of TonB fluorophores accounted for by that structure. In contrast,

TonB derivatives that dimerize in solution render their fluorophores in different

environments and yield detectable changes in the intrinsic fluorescence spectra

(100,193).

In agreement with our previous multi-cycle analyses in which TonB 33–

239 was both amine- and thiol-coupled for SPR (189), our single-cycle approach

(Figure 4.2) further demonstrates that FhuD interacts with amine-coupled TonB

33–239 with low nanomolar affinity (KD ~7 nM; Table 4.1). Our present study

also confirms that TonB 33–239 and FhuD interactions are consistent with simple

1:1 kinetics (i.e. single-cycle curve fitting with “1:1 titration” model (190); and

that binding is unaltered in the presence of siderophore. Two additional

derivatives were immobilized (TonB Δ66–100, TonB 103–239; thiol- or amine-

coupled based upon construct designs) to further delineate key regions in TonB

that mediate binding to FhuD. TonB Δ66–100 binds FhuD in a specific,

concentration-dependent manner (KD ~7.5 nM), like TonB 33–239. This result

suggests that presence of the proline-rich region in TonB (i.e. spacing between

predicted FhuD-binding regions I and II-III) is not essential for binding to FhuD.

Page 187: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

163

TonB 103–239, a derivative that still retains predicted FhuD-binding regions II

and III, also binds FhuD with low nanomolar affinity (KD ~13 nM), like TonB

33–239. This result suggests that TonB region I is not essential for complex

formation with FhuD. Within error, our SPR analyses indicate that all TonB

derivatives bind FhuD with similar affinity and in a siderophore-independent

manner. Overall, these in vitro outcomes demonstrate that residues 33–102 of

periplasmic TonB are not an absolute requirement for TonB–FhuD complex

formation.

All derivatives characterized here are elongated monomers with similar

structural features: an unstructured N-terminal region and a structured C-terminal

region. The precise location where there is crossover into structure depends on

the total length of the TonB derivative. Derivatives possessing residues 148–239

and longer exhibit C-terminal structure anywhere between residues 150–158.

This implies that there are structural differences between the predicted FhuD-

binding regions II and III. Region II is most likely disordered, given its location

within the TonB sequence. Region III is capable of adopting secondary structural

features similar to β-strand-like conformations as observed in the crystal structure

of TonB residues 148–239, the NMR structure of residues 103–239 and of TonB

bound to the transporters FhuA (96) and BtuB (97). However, region III is

disordered in smaller TonB derivatives possessing residues 155–239, indicating

that the conformation of region III depends on the presence of longer N-terminal

stretches.

Page 188: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

164

Given the flexibility of TonB, we modeled TonB-derived regions II and III

as oligopeptides with particular conformations and docked them to the more rigid

FhuD. This approach yields reasonable docking solutions for region II peptides

bound to FhuD and provides insight into the nature of TonB–FhuD interactions.

The FhuD surface to which the TonB peptide docked exhibits distinctive features.

The predicted TonB-binding surface delineates a boundary that encompasses a

landscape of varying polarity and charge (Figure 4.3B) and indicates that there are

defined clusters of TonB-binding determinants on FhuD.

The predicted TonB-binding surface also exhibits some degree of

flexibility. A region of uncertainty in the FhuD crystal structure and described by

the model’s temperature factors falls within boundaries of the predicted TonB-

binding surface (Figure 4.3C). This observation is intriguing for two reasons.

First, flexible and unstructured regions of proteins often mediate protein–protein

interactions by allowing degrees of conformational sampling necessary to

promote binding (194). Second, flexibility seems to be structurally conserved,

given that all structures of ligand-bound FhuD as well as vitamin B12-bound BtuF

are associated with high thermal parameters within this region (data not shown).

Flexibility of TonB-dependent PBPs was recently demonstrated by molecular

dynamics studies: both FhuD (122) and BtuF (121,122) undergo considerable

breathing motions, not accounted for in the crystal structures. Taken together,

these findings suggest that flexibility is also a binding determinant between TonB

and FhuD.

Page 189: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

165

Noteworthy is that the predicted TonB-binding surface of FhuD is

conserved amongst various pathogenic Gram-negative bacteria. Figure 4.4A

illustrates a sequence alignment of FhuD proteins from selected Gram-negative

bacteria. Conserved regions within this alignment are mapped onto a surface

representation of FhuD in Figure 4.4B. Conservation is visualized on the FhuD

surface through a color contour: red indicates highly conserved residues, green

indicates moderately conserved residues, and dark blue indicates non-conserved

residues. A cluster of highly conserved and moderately conserved residues falls

within the boundary delineated by the predicted TonB-binding surface.

As discussed above, this region coincides within a region of greater

flexibility and also coincides within a region of defined electrostatic and polar

properties. Taken together, these findings provide evidence that predictions borne

from phage display analyses reveal a surface on FhuD with emergent properties

that are characteristic of a protein binding site.

Results from our docking simulations reveal plausible modes for TonB

peptides that bind to FhuD. The fact that many solutions yield negative binding

energies demonstrates that favourable interactions are capable of forming even

when considering a static FhuD surface. Greater flexibility of this surface might

promote stronger interactions that were not modeled in our simulations.

Significantly, our docking solutions orient the region II oligopeptide such that the

siderophore binding site of FhuD can orient towards the OM when bound to

TonB.

Page 190: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

166

Figure 4.4. Sequence conservation of FhuD from various pathogenic Gram-negative bacteria. A.

Clustal W sequence alignment of FhuDs. Bacterial species are indicated to the left of each row in

the alignment. White letters boxed in black indicate strictly conserved residues amongst all

species. White letters boxed in dark grey indicate strongly conserved residues. Black letters

boxed in light grey indicate modestly conserved residues; B. FhuD sequence conservation mapped

to the surface of FhuD. Scores from the alignment in panel A are mapped to the surface of FhuD

and are coloured as follows: Dark blue indicates less than 20% conservation of given residues.

Green indicates approximately 50% sequence conservation and red indicates greater than 80%

sequence conservation. The predicted TonB-binding surface is outlined in black.

Despite the successes afforded from docking region II peptides to FhuD,

the same strategies were unable to yield interpretable data when region III was

docked. This outcome is probably due to incompatibility between the

conformation of region III as observed in known structures and the predicted

TonB-binding topology on FhuD. Furthermore, the co-crystal structures of TonB

bound to FhuA and to BtuB revealed that region III forms extensive interactions

Page 191: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

167

with the transporters’ Ton boxes. Accordingly, we modeled region III with a

conformation derived from these structures. However, since this conformation

yielded poor docking solutions, we can only speculate on how it could interact

with FhuD.

The distance on TonB that separates FhuD-binding region II from region

III includes nearly 20 residues (TonB residues 133–152). Because much

redundancy exists within this sequence, it probably confers little structural

propensity. However, this spacing might provide a flexible linker, enabling TonB

region III to contact more of the predicted TonB-binding surface, not greatly

populated in our docking simulations. Of the residues comprising region III, three

make hydrogen bond contacts with FhuA and BtuB Ton boxes, while four

residues project in the opposite direction. The first of these residues, TonB

residue Arg-154, could form favourable electrostatic interactions with the rest of

the FhuD surface, even when bound to an OM receptor. It is possible that our

lowest energy docking solution represents a favourable conformation of region II

bound to FhuD. TonB residues 133–152 might then act as a flexible linker that

anchors residue Arg-154 within region III to the remainder of this surface. This

orientation would place FhuD’s siderophore binding site directly beneath a

receptor’s lumen where it could capture siderophore as it emerges during

transport.

Further structural work is required to better understand the way in which

TonB and FhuD interact. Our study indicates that TonB region I is not essential

in forming a TonB–FhuD complex. While we cannot exclude the possibility that

Page 192: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

168

region I contributes in some way to complex formation, we speculate that it might

do so transiently at some point during the energy transduction cycle. Absence of

the proline-rich region also has no effect on complex formation. Similarly,

removal of the first 102 residues of TonB does not prevent complex formation.

We predict that even smaller derivatives of TonB that contain only regions II and

III will still form a complex. Such a derivative would be an ideal candidate for

structural studies as it should reduce the mobility of these N-terminal regions

when bound to FhuD. This is critical since TonB–FhuD co-crystallization

strategies may require reduction of the overall flexibility between these two

proteins. Insight provided from these approaches is necessary to reveal ways that

TonB can unify siderophore transport by acting as a protein–protein interaction

scaffold.

4.5 Acknowledgments

This work was supported by operating grants to J.W.C. from the Canadian

Institutes of Health Research (CIHR). Sheldon Biotechnology Centre is

supported by a Research Resource Grant from CIHR. Canada Foundation for

Innovation provided infrastructure for surface plasmon resonance to the Montreal

Integrated Genomics Group for Research on Infectious Pathogens. D.M.C. is

recipient of a fellowship from the Groupe d'étude des protéines membranaires,

Université de Montréal. J.C.D. is recipient of a Canada Graduate Scholarship

from the Natural Sciences and Engineering Research Council of Canada. The

authors thank C. Ng-Thow-Hing, P. Schuck and K. James for help with AUC data

collection, S.G. Paquette for providing maltose-binding protein and R. Huey and

D. Goodsell for helpful advice on conducting the docking experiments.

Page 193: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

169

Preface to Chapter 5

Determinants of binding between TonB and FhuD were identified in chapter 4.

Our finding that TonB 103–239 bound to FhuD with similar affinities as TonB 33–239

and TonB Δ66–100, suggested that it could be useful for co-crystallization trials. In

chapter 5, initial attempts of on-going efforts to co-crystallize a TonB–FhuD complex are

described. In addition, properties of TonB that are refractory to crystallization are

identified. The chapter ends with suggestions for future attempts at co-crystallizing the

complex.

Page 194: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

170

Chapter 5

Crystallization screening of the TonB–FhuD complex

Page 195: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

171

5.1 Introduction

Interactions between the energy transducer TonB, and the periplasmic

binding protein, FhuD were described in chapters 3 and 4. Both full-length

periplasmic TonB, possessing residues 33–239, and a shorter derivative

possessing residues 103–239 displayed concentration-dependent binding to FhuD,

consistent with formation of a 1:1 complex. Since TonB 103–239 possesses the

majority of predicted FhuD-binding regions and was demonstrated to be less

elongated (and presumably less flexible), it was selected as a candidate for

crystallization trials with FhuD.

To understand atomic details of TonB–FhuD interactions, structural

knowledge is desired. X-ray crystallography provides a means to directly

visualize protein–protein interactions. Determinants of binding between TonB

and FhuD can be revealed if a TonB–FhuD complex can be crystallized. A

structure solved from these crystals will directly test hypotheses borne from phage

display, biophysical, and computational analyses.

Protein crystallization remains the bottleneck of structure determination

efforts. High-throughput screening has considerably reduced the efforts required

to identify crystallization conditions. When screening, conditions that promote

protein nucleation and incorporation into a crystal lattice are identified. While

this process may directly identify conditions that generate crystals, it may also

identify promising conditions; refined screening then narrows conditions that

favour crystallization. This chapter describes efforts to purify and crystallize a

TonB–FhuD complex. In addition, the stability of TonB within the TonB–FhuD

Page 196: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

172

complex is examined. Finally, attempts to improve crystallization outcomes by

chemically cross-linking TonB and FhuD are described.

5.2 Materials and methods

5.2.1 Bacterial strains and plasmids

Decahistidine-tagged TonB 103–239 was expressed from plasmid

pDMC01, as described in chapter 4. FhuD-TEV (decahistidine-tagged FhuD plus

a TEV protease cleavage site to facilitate tag removal) was expressed from

plasmid pJD01 (chapter 4). Both were expressed in E. coli strain BL21 DE3

(pLysS). Hexahistidine-tagged TEV NIa protease was expressed from plasmid

pMHTd238, a construct from the Protein Structure Initiative Material Repository

(PSI-MR; Harvard University, Boston, MA, USA) (195). TEV was expressed in

E. coli BL21-CodonPlus-RIL (Stratagene), a strain possessing an additional

plasmid for constitutive expression of rare codons.

5.2.2 Protein expression and purification

TonB 103–239 and FhuD-TEV were expressed and purified as outlined in

chapter 4. TEV was expressed and purified as described previously (195).

5.2.3 Removal of FhuD His-tag

FhuD’s decahistidine tag was removed by TEV proteolysis. Prior to

proteolysis, FhuD-TEV was stored in chromatographic running buffer from the

Page 197: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

173

final step in its purification: 50 mM Tris pH 8.2 plus 120 mM imidazole (Fluka).

FhuD-TEV in running buffer (~20 mg) was diluted into TEV proteolysis buffer:

50 mM Tris pH 8.0, 15 mM NaCl and glycerol was added to approximately 10%

(v/v). TEV protease (~2 mg) was added to yield a TEV:FhuD ratio of 1:10 (w/w).

Cleavage proceeded overnight at room temperature with gentle stirring. Cleaved

FhuD (hereafter referred to as FhuD) was separated from TEV protease and

uncleaved FhuD by applying the reaction mixture to a Poros MC 20 Ni-chelate

column (Applied Biosystems); cleaved FhuD was collected in the flowthrough

portion. Completion of proteolysis was confirmed by SDS-PAGE and by

Western blotting for loss of immunoreactivity towards anti-His monoclonal

antibodies (Clonetech, Mountain View, CA).

5.2.4 TonB–FhuD–Fcn complex formation

Prior to complex formation, TonB 103–239 and FhuD were dialyzed

against 50 mM Tris pH 7.4, 50 mM NaCl and 10% glycerol. Protein

concentrations were determined by a Bradford Assay (Bio-Rad). To aid the

downstream strategies of structure determination, FhuD’s siderophore binding site

was saturated with the siderophore, ferricrocin (Fcn); a 10-fold molar Fcn excess

was added to FhuD. TonB–FhuD complexes were formed by mixing TonB 103–

239 with ferricrocin-bound FhuD at a 1:1 molar ratio. Complexes were

concentrated to ~10 mg/ml by ultrafiltration with YM-10 membranes (Millipore).

Equimolar complex formation was assessed by visualization of silver-stained

SDS-PAGE gels.

Page 198: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

174

5.2.5 TonB–FhuD–Fcn crystallization screening

The TonB–FhuD–Fcn complex was shipped for high-throughput

crystallization screening at the Hauptman-Woodward Medical Research Institute

(Buffalo, NY). Screening was conducted in microbatch format; distributed

among wells of a crystallization tray were 1536 different crystallization cocktail

solutions. Each well contained a different cocktail that comprised various

precipitating agents and buffers. An equal volume (< 1 µl) of the TonB–FhuD–

Fcn complex was then dispensed into each well. Paraffin oil was layered over the

wells and screening proceeded over the course of six weeks. Crystallization

outcomes were monitored weekly over the course of six weeks; magnified images

of the contents in each well were visually examined for crystallization outcomes,

or for promising leads.

5.2.6 Assessing TonB degradation

Proteolytic degradation of TonB 103–239 within the TonB–FhuD complex

and degradation of isolated TonB 103–239 were assessed by SDS-PAGE and

Western blotting. TonB 103–239 within the TonB–FhuD complex was assessed

for degradation in the absence of protease inhibitors one month after the initial

complex formation. Isolated TonB was assessed for degradation over a time

course of one week in the presence and absence of a Complete EDTA-free

protease inhibitor tablet (Roche). Degradation patterns were characterized by the

appearance of smaller molecular weight bands in a silver-stained SDS-PAGE gel

Page 199: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

175

and by loss of immunoreactivity towards anti-His monoclonal antibodies in a

Western blot.

5.2.7 TonB–FhuD cross-linking

To improve crystallizability of the TonB–FhuD complex, TonB 103–239

was cross-linked to FhuD. Proteins were dialyzed into a buffer containing 50 mM

Hepes pH 7.5 plus 150 mM NaCl. TonB and FhuD were then mixed at 1:1 molar

ratios and formaldehyde (Pierce) was added to a 10,000-fold molar excess.

Cross-linking proceeded for approximately 72 h by incubation of the reaction

mixture at 20°C without agitation. Extent of cross-linking was assessed by SDS-

PAGE and by Western blotting for immunoreactivity towards anti-His

monoclonal antibodies; samples containing 5 µg of total protein were

concentrated to dryness in a Vacufuge (Eppendorf), re-suspended in SDS-PAGE

sample buffer, and electrophoretically developed. Prior to electrophoresis,

samples were heated for 10 minutes at 60°C. As negative control, MBP was

mixed with TonB 103–239 and cross-linked as outlined above.

5.3 Results

5.3.1 Protein preparations and processing

After purification, protein purities were assessed by silver-stained SDS-

PAGE gels; all proteins purified to apparent electrophoretic homogeneity (Figure

5.1). To improve FhuD’s solubility, its decahistidine tag was removed by the

Page 200: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

176

activity of purified TEV protease. Overnight proteolysis was nearly complete and

approximately 80% of input FhuD was recovered in its cleaved form (Figure 5.1).

Furthermore, removal of FhuD’s His-tag was confirmed by loss of

immunoreactivity towards anti-His monoclonal antibodies in a Western blot (data

not shown).

5.3.2 TonB–FhuD–Fcn complex formation

Having purified both proteins to homogeneity, the TonB–FhuD complex

was formed. After saturating FhuD’s siderophore binding site with Fcn, TonB

103–239 was added to yield a complex with 1:1 molar ratio. The complex was

then concentrated to ~10 mg/ml and analyzed by SDS-PAGE to assess the

complex’s stoichiometry. When silver-stained, corresponding SDS-PAGE gels

indicated that TonB 103–239 and FhuD–Fcn were mixed at approximately the

desired molar ratio (Figure 5.2).

5.3.2 High-throughput crystallization screening

By high-throughput screening, we searched for conditions that promoted

crystallization of the TonB–FhuD–Fcn complex. After six weeks of observation,

crystal formation was not apparent. Most conditions exhibited varying degrees of

precipitation (data not shown). Other conditions failed to reveal any change and

the complex remained clarified in solution. However, a few conditions yielded

promising leads that are worthy of further investigation (Figure 5.3).

Page 201: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

177

Figure 5.1. Protein purification and processing. Typical TonB 103–239 (left) and FhuD (right)

purifications are depicted as grayscale images of silvers-stained SDS-PAGE gels. Left: TonB

103–239 preparation. Lane 1: molecular weight markers with selected bands identified; lane 2:

purified TonB 103–239. Right: FhuD preparation. Lane 1: molecular weight markers with

selected bands identified; lane 2: decahistidine-tagged FhuD; lane 3: purified TEV protease; Lane

4: cleaved, label-free FhuD.

Figure 5.2. Complexation of TonB 103–239–FhuD–Fcn. FhuD was saturated with a 10-fold

molar excess of Fcn and complexed with TonB 103–239 to yield a 1:1 protein molar ratio. A

typical TonB–FhuD complex is depicted as a grayscale image of a silver-stained SDS-PAGE gel.

Lane 1: molecular weight markers with selected weights illustrated to the left; lane 2: cleaved

FhuD standard; lane 3: TonB 103–239 standard; lanes 4–10: gradient of TonB–FhuD–Fcn

complexes (120 ng total protein in lane 4 up to 6 µg total protein in lane 10).

Page 202: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

178

Figure 5.3. Crystallization screen of the TonB 103–239–FhuD–Fcn complex. Indicated in panels

A–D are images from the most promising leads taken after six weeks of incubation. In each panel

a grayscale image is displayed to the left of an equivalent colour image. Crystallization conditions

are as follows: A. 0.1 M Ammonium sulfate, 0.1 M CAPS pH 10, 40% (w/v) PEG 4000; B. 1.9 M

Sodium malonate pH7; C. 0.12 M Sodium phosphate monobasic monohydrate, 0.68 M Potassium

phosphate dibasic pH 7.5; D. 0.1 M Sodium molybdate dihydrate, 0.1 M MES pH 6, 20% (w/v)

PEG 8000.

A

B

C

D

A

B

C

D

Page 203: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

179

Cocktails that yielded favourable outcomes exhibited no apparent trend in

composition. The most favourable precipitating agents included complex anions

such as malonate and phosphate and varying lengths of the organic polymer

polyethylene glycol (PEG). Buffer counter ions were predominantly monovalent

cations, such as Na+, and generally exhibited pH values near neutrality. However,

the buffer system compositions varied considerably and included ammonium

sulfate/CAPS, Na+/K

+ phosphate, and sodium molybdate/MES.

Outcomes of the promising conditions varied considerably. One condition

with the precipitant, PEG 4000, yielded needle-like clusters whose growth might

be controlled under favourable conditions (Figure 5.3A) Other promising

conditions yielded dense, brown-coloured particulates, the colour of which may

arise due to presence of Fcn (Figure 5.3B-D). Particulates derived from

incubation with sodium malonate, exhibited crystalline-like dimensional qualities

(Figure 5.3B). However, the small volume of this crystallization drop and the

presence of glycerol, which obscured visual inspection, made a confirmation of

crystal growth impossible.

5.3.3 TonB exhibits time-dependent degradation

Over time, TonB 103–239 suffers proteolytic cleavage leaving less in-tact

to form a complex with FhuD. After approximately two months, an

electrophoretically pure band of TonB 103–239 within the TonB–FhuD complex

(Figure 5.4A) yields a ladder of proteolytic degradation products (Figure 5.4B).

Regions within TonB that are susceptible to degradation were determined by

Page 204: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

180

Western blotting for loss of immunoreactivity towards anti-His monoclonal

antibodies. Over the course of one week and in the absence of protease inhibition,

TonB first suffers proteolytic cleavage at its N-terminus followed by loss of C-

terminal residues (Figure 5.4). Bands corresponding to the largest degradation

product are not immunoreactive towards anti-His antibodies, indicating that

TonB’s N-terminus was lost. In contrast, smaller degradation products remain

immunoreactive, indicating loss of TonB’s C-terminal residues. Addition of a

protease inhibitor cocktail improves stability. After two months, degradation was

reduced in the presence of an inhibitor cocktail tablet (data not shown).

Figure 5.4. Degradation of TonB 103–239. The susceptibility of TonB 103–239 to suffer

proteolytic degradation in complex with FhuD and in isolation was assessed by SDS-PAGE and

Western blotting. Grayscale images of silver-stained SDS-PAGE gels and a Western blot are

depicted. A. TonB–FhuD complex developed on the day it was prepared. Lane 1: molecular

weight markers. Selected weights are indicated to the left of the gel; lane 2: TonB–FhuD

complex. B. TonB–FhuD complex after 2 months. Lanes are labelled as in Panel A. C. Western

blot of isolated TonB 103–239. Lane 1: molecular weight markers. Selected weights are

indicated to the left of the blot; lane 2: TonB 103–239 after one week without proteolytic

inhibition; lane 3: silver-stained SDS-PAGE overlay of the same lane 2 material aligned to the

Western to emphasize selected loss of immunoreactivity towards anti-His monoclonal antibodies.

A B C

Page 205: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

181

5.3.4 TonB–FhuD formaldehyde-cross-linking

Formaldehyde cross-linking was conducted as a strategy to reduce

flexibility of the TonB–FhuD–Fcn complex. After 72 h, cross-linking was

evident by appearance of higher order bands that were immunoreactive towards

anti-His monoclonal antibodies in a Western blot (Figure 5.5). In the absence of

FhuD, TonB 103–239 formed an ~38 kDa cross-linked species consistent with

formation of a dimer (Figure 5.5B). In the presence of FhuD, an ~32 kDa band

appeared, in addition to the TonB dimer (Figure 5.5B). The 32 kDa band may

arise from trace amounts of uncleaved FhuD. However, it was absent in the FhuD

control and only appeared in the presence of TonB 103–239. Formation of the

TonB–FhuD complex was evident by appearance of an ~50 kDa band in the

Western (Figure 5.5B, lane 4), consistent with the molecular weight of a

Figure 5.5. Formaldehyde cross-linking of TonB–FhuD complex. Completion of cross-linking

was assessed by SDS-PAGE and Western blotting. Grayscale images of silver-stained SDS-

PAGE gels and a Western blot are depicted. A. SDS-PAGE of cross-linked samples. Lane 1:

molecular weight markers. selected weights are labelled to the left of the gel.; Lane 2: TonB 103–

239 standard; Lane 3: FhuD standard; Lane 4: TonB and FhuD mixed at a 1:1 ratio; Lane 5: MBP

standard; Lane 6: TonB and MBP mixed at a 1:1 ratio. B. Western blot of the gel displayed in

panel A. Lane identities are as in panel A. Selected molecular weights are indicated to the left of

the blot. The putative TonB–FhuD complex is marked by an arrow in lane 4.

A B

Page 206: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

182

TonB 103–239–FhuD complex (Mr = 48.1 kDa). Cross-linking was specific;

incubation with MBP failed to resolve higher order cross-linked bands in the

Western. However, cross-linking did not proceed to completion, as large amounts

of uncross-linked species were apparent in all conditions tested.

5.4 Discussion

Strategies to co-crystallize a TonB–FhuD complex require a researcher to

address many technical challenges. First, both proteins must be purified to

homogeneity. Our protein preparations display electrophoretic homogeneity and

fulfill this essential criterion. Second, proteins must remain soluble when highly

concentrated. We previously observed that FhuD is only marginally soluble when

concentrated (unpublished observations). We rationalized that removal of FhuD’s

His-tag might improve its solubility. This hypothesis proved correct. We

succeeded to complex TonB 103-239 with FhuD and concentrate the sample

enough to withstand crystallization screening without serious precipitation. After

six weeks many of the crystallization cocktails remained clarified, indicating that

the complex has no real tendency to precipitate. However, concentration of

cleaved FhuD in the absence of TonB 103–239 offers no real advantage compared

to decahistidine-tagged FhuD; both proteins precipitated with time.

A third technical challenge to overcome is protein degradation. TonB is

proteolytically labile; in the absence of protease inhibition TonB completely

degrades within two months. This is problematic for crystallization. Addition of

protease inhibitors somewhat improves the situation. However, it is unclear

Page 207: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

183

whether such additives affect crystallization outcomes. Desirable would be to

identify sites of proteolysis within TonB by mass spectrometry. Knowledge of

proteolytic sites could be used to generate stable mutants by site-directed

mutagenesis. This may stabilize TonB by obscuring protease recognition sites.

Stabilized TonB might better incorporate into a three-dimensional crystal lattice.

Discussed in chapter 4, a shorter TonB derivative that possesses only

predicted FhuD-binding regions II and III plus its C-terminal domain might be

better suited for crystallization purposes. For example, a TonB derivative

containing residues 124–239 (TonB 124–239) would possess fewer of the

residues known to be flexible and unstructured (56), yet would still retain

essential FhuD-binding regions. Those regions might be stabilized upon binding

FhuD, yielding a more stable complex.

A TonB 124–239 derivative could also be useful for crystallization

screening of a complex with ExbD. The Coulton lab possesses an ExbD construct

that expresses the entire protein as fusion to glutathione S-transferase. The

construct has been purified to homogeneity and is stable (unpublished results).

The TonB 124–239 derivative would be appropriate for complex formation with

ExbD, as it contains TonB residue 150 that was recently demonstrated to interact

with ExbD residue 92 (87).

Given that TonB 103–239 within the TonB–FhuD complex degraded with

time and is known to be conformationally flexible, we attempted to chemically

cross-link the complex. This approach was successfully used to crystallize an

adrenodoxin–adrenodoxin reductase complex (196). Our data indicated that

Page 208: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

184

TonB–FhuD complexes could be covalently trapped; however, cross-linking

never proceeded to completion. This outcome appears to be common for other

bona fide protein–protein complexes such as TonB–FepA complexes (166), and

E. coli HypB–SlyD hydrogenase accessory complexes (197). The amounts of

complex that visibly cross-linked would not yield enough material to facilitate

crystallization screening. If such amounts were purified, any unavoidable and

anticipated sample loss would yield diminishing amounts of complex.

Finally, another strategy that warrants further investigation is to increase

the molar amount of TonB 103–239 over FhuD when preparing complexes for

crystallization. This approach was successful for TonB–FhuA and TonB–BtuB

complex formation; two-fold and six-fold molar TonB excesses over FhuA and

BtuB, respectively, yielded highly diffracting crystals (96,97). Given TonB’s

proteolytic susceptibility, this approach has potential merit.

5.5 Acknowledgements

Advice and assistance with crystallization screening was provided by N.

Croteau. High-throughput screening was conducted by T. Veatch at the

Hauptman-Woodward Institute. Thanks to J. Deme for experimental

contributions.

Page 209: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

185

Chapter 6

Conclusions and future work

Page 210: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

186

6.0 Thesis objectives within the context of TonB-dependent transport

The objectives of this thesis were to characterize periplasm-localized

interactions between TonB and protein partners of the ferric hydroxamate uptake

system. In chapter 2, interactions between TonB and FhuA were predicted and

localized (189). Our use of phage display was the first literature report to

document predictions of bacterial protein–protein interactions. These predictions

were later confirmed with our TonB–FhuA crystal structure (96). In addition to

the structure’s novelty, it proved that phage display can accurately predict

protein–protein interactions. Therefore, we are confident that findings from

chapter 3, that TonB and FhuD interact (189), represent bona fide biological

interactions. Our characterization of TonB–FhuD interactions elucidated the

stoichiometry and affinity of complex formation. In chapter 4, we elucidated

regions within TonB that are essential for this interaction. We further predict that

TonB can orient FhuD towards the OM when the two proteins are complexed.

We predict that this enhances siderophore uptake by localizing FhuD near to the

inner leaflet of the OM, where it can bind siderophore during transport.

There are many reasons why this mechanism is biologically plausible. By

analogy to FepB (187), there are probably around a few thousand copies of FhuD

per cell under iron-limiting conditions. Conversely, TonB is postulated to be

present at up to 400 copies per cell (82). The affinity that we calculate and the

molar excess of FhuD over TonB would ensure that it remains bound to TonB

during transport. This feature is consistent with the postulated mechanical pulling

model of TonB-dependent energy transduction (106). By retracting towards the

Page 211: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

187

CM, TonB bound to FhuA would accomplish at least three productive outcomes.

First, retraction would cause FhuA’s cork to unfold enough to allow siderophore

translocation into the periplasm. Second, by binding and orienting FhuD towards

the OM, TonB would constructively position a siderophore binding protein.

Third, by retracting towards the CM, TonB would position siderophore-bound

FhuD nearer to the CM-embedded FhuB/C permease. This would allow FhuD to

exchange binding partners and engage its cognate permease.

Determinants of binding between TonB and FhuD govern this protein–

protein interaction. We identified features on FhuD that might comprise these

determinants. Until we elucidate the TonB–FhuD crystal structure, these features

remain speculative. However, intriguing are our findings that putative TonB-

binding determinants on FhuD are flexible. Flexibility of this FhuD region and of

TonB might act as a switch that governs binding between these proteins. Within

the context of the mechanical pulling model, TonB may conformationally cycle as

it retracts toward the CM. This could serve as a mechanism to release FhuD,

allowing delivery to FhuB/C. Reports of TonB’s conformational plasticity in vivo

support this hypothesis (79,164). Thus, we support a model whereby TonB acts

as a dynamic scaffold that couples energy transduction to protein trafficking.

Many features of this model remain to be elucidated and are now discussed.

Page 212: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

188

6.1 Directions for future research

6.1.1 Demonstration of TonB–FhuD interactions in vivo

Our biophysical characterizations have unequivocally demonstrated that

TonB and FhuD interact in vitro. Desirable would be to corroborate these

findings in vivo. Simple in vivo experimental strategies that could demonstrate

TonB–FhuD interactions include cross-linking and pull-down assays. Both

methods have benefits and drawbacks. Formaldehyde cross-linking demonstrated

TonB–FepA and TonB–ExbD interactions in vivo (87,91). However, the same

method also demonstrated TonB–OmpA and TonB–Lpp interactions, which

arguably are not biologically relevant (99).

Disulfide cross-linking might improve the specificity of interactions,

especially since we have data that demonstrates complementary regions of

interaction between TonB and FhuD. Surface-exposed residues on both proteins

that localize within these complementary regions could be individually mutated to

cysteine. Were the proteins to interact in vivo, their cross-linked species would

easily be detected by Western analysis. However, surface-exposed cysteines

might also promote homodimerization of either protein, which could interfere

with heterodimerization.

A pull-down assay could also potentially demonstrate TonB–FhuD

interactions. The experimental design would be challenging. In principle, either

histidine-tagged TonB or histidine-tagged FhuD could be immobilized on Ni2+

chelate resin as bait. If TonB were selected, it would be most desirable to capture

Page 213: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

189

its full-length, transmembrane form. However, full-length, transmembrane TonB

is both difficult to purify and prone to rapid proteolysis (unpublished

observations). Therefore, FhuD would be the logical choice as bait. Cell lysate

from a culture that expresses tagless TonB would then be incubated with

immobilized FhuD. Addition of imidazole should then elute the TonB–FhuD

complex, which could be detected by SDS-PAGE or by Western analysis.

Capture of full-length, transmembrane TonB as prey may also be confounded by

its tendency to rapidly degrade and by the membrane components of the cell

envelope.

6.1.2 Refinement of TonB–FhuD interaction localizations

Chapter 4 described interactions between TonB derivatives and FhuD.

Outcomes of these studies indicated that neither TonB’s proline-rich region, nor

residues 33-102 were essential for interaction with FhuD. Desirable, would be to

identify regions within TonB that are most essential for this interaction. Different

strategies could be employed to identify these regions. Alanine-scanning

mutagenesis could be employed to mutate TonB’s three FhuD-interacting regions

that were identified from phage display. By mutating each region, individually or

in combination, one could infer which regions are essential for interaction with

FhuD by SPR analysis. This information would guide the generation of TonB

derivatives that may be more amenable to crystallization.

Page 214: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

190

6.1.3 TonB–FhuD crystallization

Chapter 5 described our initial attempts to crystallize a TonB–FhuD

complex. Our approach favoured use of a truncated derivative of TonB and a

derivative of FhuD without its decahistidine tag. By high-throughput screening

we identified a few promising crystallization leads. These must now be refined in

order to identify conditions that grow co-crystals. The precipitants PEG and

malonate produced the most favourable outcomes. These represent the most

obvious leads to refine through further screening. Refinement of these conditions

should concentrate on screening precipitant concentrations, values of pH and

perhaps inclusion of other additives. Other possibilities were discussed in

Chapter 5.

6.1.4 Phage display predictions of TonB-interacting proteins

Phage panning against purified TonB has successfully predicted

periplasm-localized interactions between TonB and FhuA, and has successfully

predicted interactions between TonB and FhuD. Data obtained from phage

panning against purified TonB can now be used to predict additional periplasm-

localized TonB interactions. This represents an on-going effort in the Coulton lab

whereby TonB affinity-selected peptides are being compared to sequences of Fhu

system proteins: ExbB, ExbD and FhuB/C. Upon completion, these analyses

should predict all regions of interaction between TonB and partner proteins that

localize within the periplasm. This information will assist the construction of an

Page 215: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

191

interaction map that can rationalize key features of TonB-dependent energy

transduction.

6.1.5 Determination of whether TonB regulates binding of siderophores to FhuD

Our findings that TonB binds with similar affinities apo-FhuD and

siderophore-bound FhuD demonstrated that occupancy of FhuD’s binding site

does not regulate binding to TonB. However, it is still not known whether TonB

regulates binding of siderophore to FhuD. TonB-dependent regulation is possible

since our phage display analyses predicted that TonB could bind to FhuD in a

region proximal to its siderophore binding site. Whether TonB influences

siderophore binding could be determined by comparing siderophore affinities

between apo-FhuD and TonB-bound FhuD. The most direct way to accomplish

this is by titration of FhuD’s binding site with siderophore in the presence of

TonB. Fluorescence spectroscopy and isothermal titration calorimetry are two

experimental techniques to accomplish this objective. Both would enable

calculations of siderophore binding affinities.

6.1.6 Elucidation of siderophore binding sites by phage display

Our use of phage display focused on the prediction and localization of

protein–protein binding sites. However, its applications extend beyond the realm

of protein–protein interactions. Phage display has also been used to predict small

molecule binding sites on proteins (137). This application could be used to pan

phage libraries against the siderophore ferricrocin, in order to identify

Page 216: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

192

siderophore-binding patterns within the Fhu system. The known binding sites

within FhuA and FhuD would serve as excellent controls to assess the ability of

the technique to discriminate siderophore binding sites. This strategy could

potentially identify siderophore binding motifs within FhuA that could correspond

to the translocation pathway. Were such a pathway found, it could be occluded

by introduction of disulfide bonds as was previously done for FhuA (108).

Ferricrocin uptake could then be measured to see if the occlusion reduced

transport. In a similar way, ferricrocin binding sites within other siderophore-

binding proteins, such as FhuB/C could be identified. This information could also

identify ferricrocin binding sites in cytoplasmic proteins involved in siderophore

synthesis or degradation.

Page 217: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

193

References

1. Weinberg, E. D. 1997. The Lactobacillus anomaly: total iron abstinence.

Perspect. Biol. Med. 40:578-583.

2. Lieu, P. T., M. Heiskala, P. A. Peterson, and Y. Yang. 2001. The roles

of iron in health and disease. Mol. Aspects Med. 22:1-87.

3. Outten, F. W. and E. C. Theil. 2009. Iron-based Redox Switches in

Biology. Antioxid. Redox. Signal. 11:1029-1046.

4. Halliwell, B. and J. M. Gutteridge. 1984. Oxygen toxicity, oxygen

radicals, transition metals and disease. Biochem. J. 219:1-14.

5. Boukhalfa, H. and A. L. Crumbliss. 2002. Chemical aspects of

siderophore mediated iron transport. Biometals 15:325-339.

6. Raymond, K. N. and E. A. Dertz. 2004. Biochemical and physical

properties of siderophores, p. 3-17. In Jorge H.Crosa, Alexandra R.Mey,

and Shelley M.Payne (ed.), Iron Transport in Bacteria. ASM Press,

Washington D.C.

7. Raymond, K. N., E. A. Dertz, and S. S. Kim. 2003. Enterobactin: an

archetype for microbial iron transport. Proc. Natl. Acad. Sci. USA

100:3584-3588.

8. Lin, J., S. Huang, and Q. Zhang. 2002. Outer membrane proteins: key

players for bacterial adaptation in host niches. Microbes. Infect. 4:325-

331.

9. Hancock, R. E. and H. Nikaido. 1978. Outer membranes of gram-

negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1

and use in reconstitution and definition of the permeability barrier. J.

Bacteriol. 136:381-390.

10. Nikaido, H. 2003. Molecular basis of bacterial outer membrane

permeability revisited. Microbiol. Mol. Biol. Rev. 67:593-656.

11. Lerouge, I. and J. Vanderleyden. 2002. O-antigen structural variation:

mechanisms and possible roles in animal/plant-microbe interactions.

FEMS Microbiol. Rev. 26:17-47.

12. Lugtenberg, B. and A. L. Van. 1983. Molecular architecture and

functioning of the outer membrane of Escherichia coli and other gram-

negative bacteria. Biochim. Biophys. Acta 737:51-115.

Page 218: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

194

13. Nikaido, H. 1996. Outer Membrane, p. 29-47. In Escherichia coli and

Salmonella Cellular and Molecular Biology. ASM Press, Washington,

D.C.

14. Oliver, D. B. 1996. Periplasm, p. 88-103. In Escherichia coli and

Salmonella. ASM Press, Washington, D.C.

15. Parkt, J. T. 1996. The Murein Sacculus, p. 48-57. In Escherichia coli and

Salmonella: Cellular and Molecular Biology. ASM Press, Washington,

D.C.

16. Vollmer, W. and U. Bertsche. 2008. Murein (peptidoglycan) structure,

architecture and biosynthesis in Escherichia coli. Biochim. Biophys. Acta

1778:1714-1734.

17. Meroueh, S. O., K. Z. Bencze, D. Hesek, M. Lee, J. F. Fisher, T. L.

Stemmler, and S. Mobashery. 2006. Three-dimensional structure of the

bacterial cell wall peptidoglycan. Proc. Natl. Acad. Sci. USA 103:4404-

4409.

18. Gan, L., S. Chen, and G. J. Jensen. 2008. Molecular organization of

Gram-negative peptidoglycan. Proc. Natl. Acad. Sci. USA 105:18953-

18957.

19. Pautsch, A. and G. E. Schulz. 1998. Structure of the outer membrane

protein A transmembrane domain. Nat. Struct. Biol. 5:1013-1017.

20. Ni, Y., J. Reye, and R. R. Chen. 2007. Lpp deletion as a permeabilization

method. Biotechnol. Bioeng. 97:1347-1356.

21. Shu, W., J. Liu, H. Ji, and M. Lu. 2000. Core structure of the outer

membrane lipoprotein from Escherichia coli at 1.9 Å resolution. J. Mol.

Biol. 299:1101-1112.

22. Piovant, M. and C. Lazdunski. 1982. Biosynthesis of Escherichia coli

Braun's lipoprotein precursors in vitro and binding to membrane vesicles.

Eur. J. Biochem. 125:623-629.

23. Cascales, E., A. Bernadac, M. Gavioli, J. C. Lazzaroni, and R.

Lloubes. 2002. Pal lipoprotein of Escherichia coli plays a major role in

outer membrane integrity. J. Bacteriol. 184:754-759.

24. Kadner, R. J. 1996. Cytoplasmic Membrane, p. 58-87. In Escherichia

coli and Salmonella Cellular and Molecular Biology. ASM Press,

Washington D.C.

Page 219: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

195

25. Basle, A., G. Rummel, P. Storici, J. P. Rosenbusch, and T. Schirmer.

2006. Crystal structure of osmoporin OmpC from E. coli at 2.0 Å. J. Mol.

Biol. 362:933-942.

26. Cowan, S. W., T. Schirmer, G. Rummel, M. Steiert, R. Ghosh, R. A.

Pauptit, J. N. Jansonius, and J. P. Rosenbusch. 1992. Crystal structures

explain functional properties of two E. coli porins. Nature 358:727-733.

27. Yamashita, E., M. V. Zhalnina, S. D. Zakharov, O. Sharma, and W.

A. Cramer. 2008. Crystal structures of the OmpF porin: function in a

colicin translocon. EMBO J. 27:2171-2180.

28. Perozo, E. and D. C. Rees. 2003. Structure and mechanism in prokaryotic

mechanosensitive channels. Curr. Opin. Struct. Biol. 13:432-442.

29. Dutzler, R., Y. F. Wang, P. Rizkallah, J. P. Rosenbusch, and T.

Schirmer. 1996. Crystal structures of various maltooligosaccharides

bound to maltoporin reveal a specific sugar translocation pathway.

Structure. 4:127-134.

30. Charbit, A. 2003. Maltodextrin transport through LamB. Front Biosci.

8:265-274.

31. Hearn, E. M., D. R. Patel, B. W. Lepore, M. Indic, and B. van den

Berg. 2009. Transmembrane passage of hydrophobic compounds through

a protein channel wall. Nature 458:367-370.

32. Stojiljkovic, I., M. Cobeljic, and K. Hantke. 1993. Escherichia coli K-

12 ferrous iron uptake mutants are impaired in their ability to colonize the

mouse intestine. FEMS Microbiol. Lett. 108:111-115.

33. Cartron, M. L., S. Maddocks, P. Gillingham, C. J. Craven, and S. C.

Andrews. 2006. Feo--transport of ferrous iron into bacteria. Biometals

19:143-157.

34. Blanton, K. J., G. D. Biswas, J. Tsai, J. Adams, D. W. Dyer, S. M.

Davis, G. G. Koch, P. K. Sen, and P. F. Sparling. 1990. Genetic

evidence that Neisseria gonorrhoeae produces specific receptors for

transferrin and lactoferrin. J. Bacteriol. 172:5225-5235.

35. Legrain, M., V. Mazarin, S. W. Irwin, B. Bouchon, M. J. Quentin-

Millet, E. Jacobs, and A. B. Schryvers. 1993. Cloning and

characterization of Neisseria meningitidis genes encoding the transferrin-

binding proteins Tbp1 and Tbp2. Gene 130:73-80.

36. Dhaenens, L., F. Szczebara, and M. O. Husson. 1997. Identification,

characterization, and immunogenicity of the lactoferrin-binding protein

from Helicobacter pylori. Infect. Immun. 65:514-518.

Page 220: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

196

37. Cescau, S., H. Cwerman, S. Letoffe, P. Delepelaire, C. Wandersman,

and F. Biville. 2007. Heme acquisition by hemophores. Biometals 20:603-

613.

38. Crosa, J. H. and C. T. Walsh. 2002. Genetics and assembly line

enzymology of siderophore biosynthesis in bacteria. Microbiol. Mol. Biol.

Rev. 66:223-249.

39. Jones, A. M., S. E. Lindow, and M. C. Wildermuth. 2007. Salicylic

acid, yersiniabactin, and pyoverdin production by the model

phytopathogen Pseudomonas syringae pv. tomato DC3000: synthesis,

regulation, and impact on tomato and Arabidopsis host plants. J. Bacteriol.

189:6773-6786.

40. Miethke, M. and M. A. Marahiel. 2007. Siderophore-based iron

acquisition and pathogen control. Microbiol. Mol. Biol. Rev. 71:413-451.

41. Braun, V. 2009. FhuA (TonA), the career of a protein. J. Bacteriol.

191:3431-3436.

42. Ferguson, A. D., E. Hofmann, J. W. Coulton, K. Diederichs, and W.

Welte. 1998. Siderophore-mediated iron transport: crystal structure of

FhuA with bound lipopolysaccharide. Science 282:2215-2220.

43. Ferguson, A. D., W. Welte, E. Hofmann, B. Lindner, O. Holst, J. W.

Coulton, and K. Diederichs. 2000. A conserved structural motif for

lipopolysaccharide recognition by procaryotic and eucaryotic proteins.

Structure. 8:585-592.

44. Buchanan, S. K., B. S. Smith, L. Venkatramani, D. Xia, L. Esser, M.

Palnitkar, R. Chakraborty, D. van der Helm, and J. Deisenhofer.

1999. Crystal structure of the outer membrane active transporter FepA

from Escherichia coli. Nat. Struct. Biol. 6:56-63.

45. Ferguson, A. D., R. Chakraborty, B. S. Smith, L. Esser, D. van der

Helm, and J. Deisenhofer. 2002. Structural basis of gating by the outer

membrane transporter FecA. Science 295:1715-1719.

46. Chimento, D. P., A. K. Mohanty, R. J. Kadner, and M. C. Wiener.

2003. Substrate-induced transmembrane signaling in the cobalamin

transporter BtuB. Nat. Struct. Biol. 10:394-401.

47. Sharma, O., E. Yamashita, M. V. Zhalnina, S. D. Zakharov, K. A.

Datsenko, B. L. Wanner, and W. A. Cramer. 2007. Structure of the

complex of the colicin E2 R-domain and its BtuB receptor. The outer

membrane colicin translocon. J. Biol. Chem. 282:23163-23170.

Page 221: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

197

48. Cherezov, V., E. Yamashita, W. Liu, M. Zhalnina, W. A. Cramer, and

M. Caffrey. 2006. In meso structure of the cobalamin transporter, BtuB, at

1.95 Å resolution. J. Mol. Biol. 364:716-734.

49. Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova,

T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer.

2003. The structure of BtuB with bound colicin E3 R-domain implies a

translocon. Nat. Struct. Biol. 10:948-954.

50. Buchanan, S. K., P. Lukacik, S. Grizot, R. Ghirlando, M. M. Ali, T. J.

Barnard, K. S. Jakes, P. K. Kienker, and L. Esser. 2007. Structure of

colicin I receptor bound to the R-domain of colicin Ia: implications for

protein import. EMBO J. 26:2594-2604.

51. Cobessi, D., H. Celia, and F. Pattus. 2005. Crystal structure at high

resolution of ferric-pyochelin and its membrane receptor FptA from

Pseudomonas aeruginosa. J. Mol. Biol. 352:893-904.

52. Cobessi, D., H. Celia, N. Folschweiller, I. J. Schalk, M. A. Abdallah,

and F. Pattus. 2005. The crystal structure of the pyoverdine outer

membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms

resolution. J. Mol. Biol. 347:121-134.

53. Krieg, S., F. Huche, K. Diederichs, N. Izadi-Pruneyre, A. Lecroisey, C.

Wandersman, P. Delepelaire, and W. Welte. 2009. Heme uptake across

the outer membrane as revealed by crystal structures of the receptor-

hemophore complex. Proc. Natl. Acad. Sci. USA 106:1045-1050.

54. Brillet, K., A. Meksem, E. Lauber, C. Reimmann, and D. Cobessi.

2009. Use of an in-house approach to study the three-dimensional

structures of various outer membrane proteins: structure of the alcaligin

outer membrane transporter FauA from Bordetella pertussis. Acta

Crystallogr. D. Biol. Crystallogr. 65:326-331.

55. Gudmundsdottir, A., P. E. Bell, M. D. Lundrigan, C. Bradbeer, and R.

J. Kadner. 1989. Point mutations in a conserved region (TonB box) of

Escherichia coli outer membrane protein BtuB affect vitamin B12

transport. J. Bacteriol. 171:6526-6533.

56. Peacock, R. S., V. V. Andrushchenko, A. R. Demcoe, M. Gehmlich, L.

S. Lu, A. G. Herrero, and H. J. Vogel. 2006. Characterization of TonB

interactions with the FepA cork domain and FecA N-terminal signaling

domain. Biometals 19:127-142.

57. Ferguson, A. D., C. A. Amezcua, N. M. Halabi, Y. Chelliah, M. K.

Rosen, R. Ranganathan, and J. Deisenhofer. 2007. Signal transduction

pathway of TonB-dependent transporters. Proc. Natl. Acad. Sci. USA

104:513-518.

Page 222: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

198

58. Brillet, K., L. Journet, H. Celia, L. Paulus, A. Stahl, F. Pattus, and D.

Cobessi. 2007. A beta strand lock exchange for signal transduction in

TonB-dependent transducers on the basis of a common structural motif.

Structure. 15:1383-1391.

59. Fanucci, G. E., J. Y. Lee, and D. S. Cafiso. 2003. Spectroscopic

evidence that osmolytes used in crystallization buffers inhibit a

conformation change in a membrane protein. Biochemistry 42:13106-

13112.

60. Kim, M., G. E. Fanucci, and D. S. Cafiso. 2007. Substrate-dependent

transmembrane signaling in TonB-dependent transporters is not

conserved. Proc. Natl. Acad. Sci. USA 104:11975-11980.

61. Ferguson, A. D., V. Braun, H. P. Fiedler, J. W. Coulton, K.

Diederichs, and W. Welte. 2000. Crystal structure of the antibiotic

albomycin in complex with the outer membrane transporter FhuA. Protein

Sci. 9:956-963.

62. Ferguson, A. D., J. Ködding, G. Walker, C. Bos, J. W. Coulton, K.

Diederichs, V. Braun, and W. Welte. 2001. Active transport of an

antibiotic rifamycin derivative by the outer-membrane protein FhuA.

Structure. 9:707-716.

63. Yue, W. W., S. Grizot, and S. K. Buchanan. 2003. Structural evidence

for iron-free citrate and ferric citrate binding to the TonB-dependent outer

membrane transporter FecA. J. Mol. Biol. 332:353-368.

64. Xu, Q., J. F. Ellena, M. Kim, and D. S. Cafiso. 2006. Substrate-

dependent unfolding of the energy coupling motif of a membrane transport

protein determined by double electron-electron resonance. Biochemistry

45:10847-10854.

65. Blanvillain, S., D. Meyer, A. Boulanger, M. Lautier, C. Guynet, N.

Denancé, J. Vasse, E. Lauber, and M. Arlat. 2007. Plant carbohydrate

scavenging through tonb-dependent receptors: a feature shared by

phytopathogenic and aquatic bacteria. PLoS. One. 2:e224.

66. Schauer, K., B. Gouget, M. Carrière, A. Labigne, and H. de Reuse.

2007. Novel nickel transport mechanism across the bacterial outer

membrane energized by the TonB/ExbB/ExbD machinery. Mol.

Microbiol. 63:1054-1068.

67. Tralau, T., S. Vuilleumier, C. Thibault, B. J. Campbell, C. A. Hart,

and M. A. Kertesz. 2007. Transcriptomic analysis of the sulfate

starvation response of Pseudomonas aeruginosa. J. Bacteriol. 189:6743-

6750.

Page 223: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

199

68. Chu, B. C., R. S. Peacock, and H. J. Vogel. 2007. Bioinformatic analysis

of the TonB protein family. Biometals 20:467-483.

69. Evans, J. S., B. A. Levine, I. P. Trayer, C. J. Dorman, and C. F.

Higgins. 1986. Sequence-imposed structural constraints in the TonB

protein of E. coli. FEBS Lett. 208:211-216.

70. Brewer, S., M. Tolley, I. P. Trayer, G. C. Barr, C. J. Dorman, K.

Hannavy, C. F. Higgins, J. S. Evans, B. A. Levine, and M. R.

Wormald. 1990. Structure and function of X-Pro dipeptide repeats in the

TonB proteins of Salmonella typhimurium and Escherichia coli. J. Mol.

Biol. 216:883-895.

71. Larsen, R. A., G. E. Wood, and K. Postle. 1994. The conserved proline-

rich motif is not essential for energy transduction by Escherichia coli

TonB protein. Mol. Microbiol. 12:857.

72. Chang, C., A. Mooser, A. Plückthun, and A. Wlodawer. 2001. Crystal

structure of the dimeric C-terminal domain of TonB reveals a novel fold.

J. Biol. Chem. 276:27535-27540.

73. Koedding, J., P. Howard, L. Kaufmann, P. Polzer, A. Lustig, and W.

Welte. 2004. Dimerization of TonB is not essential for its binding to the

outer membrane siderophore receptor FhuA of Escherichia coli. J. Biol.

Chem. 279:9978-9986.

74. Ködding, J. F., F. Killig, P. Polzer, S. P. Howard, K. Diederichs, and

W. Welte. 2005. Crystal structure of a 92-residue C-terminal fragment of

TonB from Escherichia coli reveals significant conformational changes

compared to structures of smaller TonB fragments. J. Biol. Chem.

280:3022-3028.

75. Peacock, R. S., A. M. Weljie, S. P. Howard, F. D. Price, and H. J.

Vogel. 2005. The solution structure of the C-terminal domain of TonB and

interaction studies with TonB box peptides. J. Mol. Biol. 345:1185-1197.

76. Lopez, C. S., R. S. Peacock, J. H. Crosa, and H. J. Vogel. 2009.

Molecular characterization of the TonB2 protein from the fish pathogen

Vibrio anguillarum. Biochem. J. 418:49-59.

77. Khursigara, C. M., G. De Crescenzo, P. D. Pawelek, and J. W.

Coulton. 2004. Enhanced binding of TonB to a ligand-loaded outer

membrane receptor: role of the oligomeric state of TonB in formation of a

functional FhuA•TonB complex. J. Biol. Chem. 279:7405-7412.

78. Sauter, A., S. P. Howard, and V. Braun. 2003. In vivo evidence for

TonB dimerization. J. Bacteriol. 185:5747-5754.

Page 224: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

200

79. Ghosh, J. and K. Postle. 2005. Disulphide trapping of an in vivo energy-

dependent conformation of Escherichia coli TonB protein. Mol.

Microbiol. 55:276-288.

80. Kampfenkel, K. and V. Braun. 1993. Topology of the ExbB protein in

the cytoplasmic membrane of Escherichia coli. J. Biol. Chem. 268:6050-

6057.

81. Garcia-Herrero, A., R. S. Peacock, S. P. Howard, and H. J. Vogel.

2007. The solution structure of the periplasmic domain of the TonB

system ExbD protein reveals an unexpected structural homology with

siderophore-binding proteins. Mol. Microbiol. 66:872-889.

82. Higgs, P. I., R. A. Larsen, and K. Postle. 2002. Quantification of known

components of the Escherichia coli TonB energy transduction system:

TonB, ExbB, ExbD and FepA. Mol. Microbiol. 44:271-281.

83. Zhai, Y. F., W. Heijne, and M. H. Saier, Jr. 2003. Molecular modeling

of the bacterial outer membrane receptor energizer, ExbBD/TonB, based

on homology with the flagellar motor, MotAB. Biochim. Biophys. Acta

1614:201-210.

84. Braun, V. and C. Herrmann. 2004. Point mutations in transmembrane

helices 2 and 3 of ExbB and TolQ affect their activities in Escherichia coli

K-12. J. Bacteriol. 186:4402-4406.

85. Larsen, R. A. and K. Postle. 2001. Conserved residues Ser(16) and

His(20) and their relative positioning are essential for TonB activity,

cross-linking of TonB with ExbB, and the ability of TonB to respond to

proton motive force. J. Biol. Chem. 276:8111-8117.

86. Larsen, R. A., G. E. Deckert, K. A. Kastead, S. Devanathan, K. L.

Keller, and K. Postle. 2007. His(20) provides the sole functionally

significant side chain in the essential TonB transmembrane domain. J.

Bacteriol. 189:2825-2833.

87. Ollis, A. A., M. Manning, K. G. Held, and K. Postle. 2009. Cytoplasmic

membrane proton motive force energizes periplasmic interactions between

ExbD and TonB. Mol. Microbiol. 73:466-481.

88. Gunter, K. and V. Braun. 1990. In vivo evidence for FhuA outer

membrane receptor interaction with the TonB inner membrane protein of

Escherichia coli. FEBS Lett. 274:85-88.

89. Moeck, G. S., J. W. Coulton, and K. Postle. 1997. Cell envelope

signaling in Escherichia coli. Ligand binding to the ferrichrome-iron

receptor fhua promotes interaction with the energy-transducing protein

TonB. J. Biol. Chem. 272:28391-28397.

Page 225: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

201

90. Skare, J. T., B. M. Ahmer, C. L. Seachord, R. P. Darveau, and K.

Postle. 1993. Energy transduction between membranes. TonB, a

cytoplasmic membrane protein, can be chemically cross-linked in vivo to

the outer membrane receptor FepA. J. Biol. Chem. 268:16302-16308.

91. Larsen, R. A., D. Foster-Hartnett, M. A. McIntosh, and K. Postle.

1997. Regions of Escherichia coli TonB and FepA proteins essential for in

vivo physical interactions. J. Bacteriol. 179:3213-3221.

92. Cadieux, N., C. Bradbeer, and R. J. Kadner. 2000. Sequence changes in

the ton box region of BtuB affect its transport activities and interaction

with TonB protein. J. Bacteriol. 182:5954-5961.

93. Cadieux, N. and R. J. Kadner. 1999. Site-directed disulfide bonding

reveals an interaction site between energy-coupling protein TonB and

BtuB, the outer membrane cobalamin transporter. Proc. Natl. Acad. Sci.

USA 96:10673-10678.

94. Khursigara, C. M., G. De Crescenzo, P. D. Pawelek, and J. W.

Coulton. 2005. Deletion of the proline-rich region of TonB disrupts

formation of a 2:1 complex with FhuA, an outer membrane receptor of

Escherichia coli. Protein Sci. 14:1266-1273.

95. Khursigara, C. M., G. De Crescenzo, P. D. Pawelek, and J. W.

Coulton. 2005. Kinetic analyses reveal multiple steps in forming TonB-

FhuA complexes from Escherichia coli. Biochemistry 44:3441-3453.

96. Pawelek, P. D., N. Croteau, C. Ng-Thow-Hing, C. M. Khursigara, N.

Moiseeva, M. Allaire, and J. W. Coulton. 2006. Structure of TonB in

complex with FhuA, E. coli outer membrane receptor. Science 312:1399-

1402.

97. Shultis, D. D., M. D. Purdy, C. N. Banchs, and M. C. Wiener. 2006.

Outer membrane active transport: structure of the BtuB:TonB complex.

Science 312:1396-1399.

98. James, K. J., M. A. Hancock, V. Moreau, F. Molina, and J. W.

Coulton. 2008. TonB induces conformational changes in surface-exposed

loops of FhuA, outer membrane receptor of Escherichia coli. Protein Sci.

17:1679-1688.

99. Higgs, P. I., T. E. Letain, K. K. Merriam, N. S. Burke, H. Park, C.

Kang, and K. Postle. 2002. TonB interacts with nonreceptor proteins in

the outer membrane of Escherichia coli. J. Bacteriol. 184:1640-1648.

100. Kaserer, W. A., X. Jiang, Q. Xiao, D. C. Scott, M. Bauler, D.

Copeland, S. M. Newton, and P. E. Klebba. 2008. Insight from TonB

Page 226: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

202

hybrid proteins into the mechanism of iron transport through the outer

membrane. J. Bacteriol. 190:4001-4016.

101. Letain, T. E. and K. Postle. 1997. TonB protein appears to transduce

energy by shuttling between the cytoplasmic membrane and the outer

membrane in Escherichia coli. Mol. Microbiol. 24:271-283.

102. Larsen, R. A., T. E. Letain, and K. Postle. 2003. In vivo evidence of

TonB shuttling between the cytoplasmic and outer membrane in

Escherichia coli. Mol. Microbiol. 49:211-218.

103. Feilmeier, B. J., G. Iseminger, D. Schroeder, H. Webber, and G. J.

Phillips. 2000. Green fluorescent protein functions as a reporter for

protein localization in Escherichia coli. J. Bacteriol. 182:4068-4076.

104. Chimento, D. P., R. J. Kadner, and M. C. Wiener. 2005. Comparative

structural analysis of TonB-dependent outer membrane transporters:

implications for the transport cycle. Proteins 59:240-251.

105. Brockwell, D. J., E. Paci, R. C. Zinober, G. S. Beddard, P. D. Olmsted,

D. A. Smith, R. N. Perham, and S. E. Radford. 2003. Pulling geometry

defines the mechanical resistance of a beta-sheet protein. Nat. Struct. Biol.

10:731-737.

106. Gumbart, J., M. C. Wiener, and E. Tajkhorshid. 2007. Mechanics of

force propagation in TonB-dependent outer membrane transport. Biophys.

J. 93:496-504.

107. Chakraborty, R., E. Storey, and D. van der Helm. 2007. Molecular

mechanism of ferricsiderophore passage through the outer membrane

receptor proteins of Escherichia coli. Biometals 20:263-274.

108. Eisenhauer, H. A., S. Shames, P. D. Pawelek, and J. W. Coulton. 2005.

Siderophore transport through Escherichia coli outer membrane receptor

FhuA with disulfide-tethered cork and barrel domains. J. Biol. Chem.

280:30574-30580.

109. Devanathan, S. and K. Postle. 2007. Studies on colicin B translocation:

FepA is gated by TonB. Mol. Microbiol. 65:441-453.

110. Ma, L., W. Kaserer, R. Annamalai, D. C. Scott, B. Jin, X. Jiang, Q.

Xiao, H. Maymani, L. M. Massis, L. C. Ferreira, S. M. Newton, and P.

E. Klebba. 2007. Evidence of ball-and-chain transport of ferric

enterobactin through FepA. J. Biol. Chem. 282:397-406.

111. Clarke, T. E., S. Y. Ku, D. R. Dougan, H. J. Vogel, and L. W. Tari.

2000. The structure of the ferric siderophore binding protein FhuD

complexed with gallichrome. Nat. Struct. Biol. 7:287-291.

Page 227: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

203

112. Clarke, T. E., V. Braun, G. Winkelmann, L. W. Tari, and H. J. Vogel.

2002. X-ray crystallographic structures of the Escherichia coli periplasmic

protein FhuD bound to hydroxamate-type siderophores and the antibiotic

albomycin. J. Biol. Chem. 277:13966-13972.

113. Rohrbach, M. R., V. Braun, and W. Köster. 1995. Ferrichrome

transport in Escherichia coli K-12: altered substrate specificity of mutated

periplasmic FhuD and interaction of FhuD with the integral membrane

protein FhuB. J. Bacteriol. 177:7186-7193.

114. Borths, E. L., K. P. Locher, A. T. Lee, and D. C. Rees. 2002. The

structure of Escherichia coli BtuF and binding to its cognate ATP binding

cassette transporter. Proc. Natl. Acad. Sci. U. S. A 99:16642-16647.

115. Cadieux, N., C. Bradbeer, E. Reeger-Schneider, W. Köster, A. K.

Mohanty, M. C. Wiener, and R. J. Kadner. 2002. Identification of the

periplasmic cobalamin-binding protein BtuF of Escherichia coli. J.

Bacteriol. 184:706-717.

116. Boos, W. and H. Shuman. 1998. Maltose/maltodextrin system of

Escherichia coli: transport, metabolism, and regulation. Microbiol. Mol.

Biol. Rev. 62:204-229.

117. Quiocho, F. A., J. C. Spurlino, and L. E. Rodseth. 1997. Extensive

features of tight oligosaccharide binding revealed in high-resolution

structures of the maltodextrin transport/chemosensory receptor. Structure.

5:997-1015.

118. Telmer, P. G. and B. H. Shilton. 2003. Insights into the conformational

equilibria of maltose-binding protein by analysis of high affinity mutants.

J. Biol. Chem. 278:34555-34567.

119. Krewulak, K. D., C. M. Shepherd, and H. J. Vogel. 2005. Molecular

dynamics simulations of the periplasmic ferric-hydroxamate binding

protein FhuD. Biometals 18:375-386.

120. Karpowich, N. K., H. H. Huang, P. C. Smith, and J. F. Hunt. 2003.

Crystal structures of the BtuF periplasmic-binding protein for vitamin B12

suggest a functionally important reduction in protein mobility upon ligand

binding. J. Biol. Chem. 278:8429-8434.

121. Liu, M., T. Sun, J. Hu, W. Chen, and C. Wang. 2008. Study on the

mechanism of the BtuF periplasmic-binding protein for vitamin B12.

Biophys. Chem. 135:19-24.

122. Kandt, C., Z. Xu, and D. P. Tieleman. 2006. Opening and closing

motions in the periplasmic vitamin B12 binding protein BtuF.

Biochemistry 45:13284-13292.

Page 228: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

204

123. Shi, R., A. Proteau, J. Wagner, Q. Cui, E. O. Purisima, A. Matte, and

M. Cygler. 2009. Trapping open and closed forms of FitE: a group III

periplasmic binding protein. Proteins 75:598-609.

124. Procko, E., M. L. O'Mara, W. F. Bennett, D. P. Tieleman, and R.

Gaudet. 2009. The mechanism of ABC transporters: general lessons from

structural and functional studies of an antigenic peptide transporter.

FASEB J. 23:1287-1302.

125. Krewulak, K. D. and H. J. Vogel. 2008. Structural biology of bacterial

iron uptake. Biochim. Biophys. Acta 1778:1781-1804.

126. Locher, K. P., A. T. Lee, and D. C. Rees. 2002. The E. coli BtuCD

structure: a framework for ABC transporter architecture and mechanism.

Science 296:1091-1098.

127. Pinkett, H. W., A. T. Lee, P. Lum, K. P. Locher, and D. C. Rees. 2007.

An inward-facing conformation of a putative metal-chelate-type ABC

transporter. Science 315:373-377.

128. Hollenstein, K., D. C. Frei, and K. P. Locher. 2007. Structure of an

ABC transporter in complex with its binding protein. Nature 446:213-216.

129. Oldham, M. L., D. Khare, F. A. Quiocho, A. L. Davidson, and J. Chen.

2007. Crystal structure of a catalytic intermediate of the maltose

transporter. Nature 450:515-521.

130. Hvorup, R. N., B. A. Goetz, M. Niederer, K. Hollenstein, E. Perozo,

and K. P. Locher. 2007. Asymmetry in the structure of the ABC

transporter-binding protein complex BtuCD-BtuF. Science 317:1387-

1390.

131. Sebulsky, M. T., B. H. Shilton, C. D. Speziali, and D. E. Heinrichs.

2003. The role of FhuD2 in iron(III)-hydroxamate transport in

Staphylococcus aureus. Demonstration that FhuD2 binds iron(III)-

hydroxamates but with minimal conformational change and implication of

mutations on transport. J. Biol. Chem. 278:49890-49900.

132. Mademidis, A., H. Killmann, W. Kraas, I. Flechsler, G. Jung, and V.

Braun. 1997. ATP-dependent ferric hydroxamate transport system in

Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a

transmembrane/cytoplasmic region of the integral membrane protein

FhuB, as revealed by competitive peptide mapping. Mol. Microbiol.

26:1109-1123.

133. Locher, K. P. 2009. Structure and mechanism of ATP-binding cassette

transporters. Philos. Trans. R. Soc. Lond B Biol. Sci. 364:239-245.

Page 229: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

205

134. Greenwood, K. T. and R. K. Luke. 1978. Enzymatic hydrolysis of

enterochelin and its iron complex in Escherichia Coli K-12. Properties of

enterochelin esterase. Biochim. Biophys. Acta 525:209-218.

135. Matzanke, B. F., S. Anemuller, V. Schunemann, A. X. Trautwein, and

K. Hantke. 2004. FhuF, part of a siderophore-reductase system.

Biochemistry 43:1386-1392.

136. Expert, D., A. Boughammoura, and T. Franza. 2008. Siderophore-

controlled iron assimilation in the enterobacterium Erwinia chrysanthemi:

evidence for the involvement of bacterioferritin and the Suf iron-sulfur

cluster assembly machinery. J. Biol. Chem. 283:36564-36572.

137. Rodi, D. J., R. W. Janes, H. J. Sanganee, R. A. Holton, B. A. Wallace,

and L. Makowski. 1999. Screening of a library of phage-displayed

peptides identifies human bcl-2 as a taxol-binding protein. J. Mol. Biol.

285:197-203.

138. Rodi, D. J., G. E. Agoston, R. Manon, R. Lapcevich, S. J. Green, and

L. Makowski. 2001. Identification of small molecule binding sites within

proteins using phage display technology. Comb. Chem. High Throughput.

Screen. 4:553-572.

139. Burch, L. R., M. Scott, E. Pohler, D. Meek, and T. Hupp. 2004. Phage-

peptide display identifies the interferon-responsive, death-activated protein

kinase family as a novel modifier of MDM2 and p21WAF1. J. Mol. Biol.

337:115-128.

140. Rodi, D. J., A. S. Soares, and L. Makowski. 2002. Quantitative

assessment of peptide sequence diversity in M13 combinatorial peptide

phage display libraries. J. Mol. Biol. 322:1039-1052.

141. Piliarik, M., H. Vaisocherova, and J. Homola. 2009. Surface plasmon

resonance biosensing. Methods Mol. Biol. 503:65-88.

142. Philo, J. S. 2006. Is any measurement method optimal for all aggregate

sizes and types? AAPS. J. 8:E564-E571.

143. Schuck, P. 2000. Size-distribution analysis of macromolecules by

sedimentation velocity ultracentrifugation and Lamm equation modeling.

Biophys. J. 78:1606-1619.

144. Schuck, P. 1998. Sedimentation analysis of noninteracting and self-

associating solutes using numerical solutions to the Lamm equation.

Biophys. J. 75:1503-1512.

145. Wandersman, C. and P. Delepelaire. 2004. Bacterial iron sources: from

siderophores to hemophores. Annu. Rev. Microbiol. 58:611-647.

Page 230: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

206

146. Braun, V. and M. Braun. 2002. Iron transport and signaling in

Escherichia coli. FEBS Lett. 529:78-85.

147. Braun, V., K. Hantke, and W. Köster. 1998. Bacterial Iron Transport:

Mechanisms, Genetics, and Regulation., p. 68-145. In Metal ions in

biological systems. Marcel Dekker Inc., New York.

148. Ferguson, A. D., J. W. Coulton, K. Diederichs, and W. Welte. 2001.

The ferric hydroxamate uptake receptor FhuA and related TonB-

dependent transporters in the outer membrane of gram-negative bacteria.,

p. 834-849. In Handbook of Metalloproteins. John Wiley & Sons Ltd.,

Chichester.

149. Moeck, G. S. and J. W. Coulton. 1998. TonB-dependent iron acquisition:

mechanisms of siderophore-mediated active transport. Mol. Microbiol.

28:675-681.

150. Locher, K. P., B. Rees, R. Koebnik, A. Mitschler, L. Moulinier, J. P.

Rosenbusch, and D. Moras. 1998. Transmembrane signaling across the

ligand-gated FhuA receptor: crystal structures of free and ferrichrome-

bound states reveal allosteric changes. Cell 95:771-778.

151. Faraldo-Gómez, J. D., G. R. Smith, and M. S. Sansom. 2003.

Molecular dynamics simulations of the bacterial outer membrane protein

FhuA: a comparative study of the ferrichrome-free and bound states.

Biophys. J. 85:1406-1420.

152. Postle, K. and R. J. Kadner. 2003. Touch and go: tying TonB to

transport. Mol. Microbiol. 49:869-882.

153. Kadner, R. J. 1990. Vitamin B12 transport in Escherichia coli: energy

coupling between membranes. Mol. Microbiol. 4:2027-2033.

154. Schöffler, H. and V. Braun. 1989. Transport across the outer membrane

of Escherichia coli K-12 via the FhuA receptor is regulated by the TonB

protein of the cytoplasmic membrane. Mol. Gen. Genet. 217:378-383.

155. Ogierman, M. and V. Braun. 2003. Interactions between the outer

membrane ferric citrate transporter FecA and TonB: studies of the FecA

TonB box. J. Bacteriol. 185:1870-1885.

156. Merianos, H. J., N. Cadieux, C. H. Lin, R. J. Kadner, and D. S. Cafiso.

2000. Substrate-induced exposure of an energy-coupling motif of a

membrane transporter. Nat. Struct. Biol. 7:205-209.

157. Cadieux, N., P. G. Phan, D. S. Cafiso, and R. J. Kadner. 2003.

Differential substrate-induced signaling through the TonB-dependent

transporter BtuB. Proc. Natl. Acad. Sci. USA 100:10688-10693.

Page 231: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

207

158. Braun, M., H. Killmann, and V. Braun. 1999. The -barrel domain of

FhuA5-160 is sufficient for TonB-dependent FhuA activities of

Escherichia coli. Mol. Microbiol. 33:1037-1049.

159. Scott, D. C., Z. Cao, Z. Qi, M. Bauler, J. D. Igo, S. M. Newton, and P.

E. Klebba. 2001. Exchangeability of N termini in the ligand-gated porins

of Escherichia coli. J. Biol. Chem. 276:13025-13033.

160. Howard, S. P., C. Herrmann, C. W. Stratilo, and V. Braun. 2001. In

vivo synthesis of the periplasmic domain of TonB inhibits transport

through the FecA and FhuA iron siderophore transporters of Escherichia

coli. J. Bacteriol. 183:5885-5895.

161. Killmann, H., C. Herrmann, A. Torun, G. Jung, and V. Braun. 2002.

TonB of Escherichia coli activates FhuA through interaction with the -

barrel. Microbiology 148:3497-3509.

162. Vakharia, H. L. and K. Postle. 2002. FepA with globular domain

deletions lacks activity. J. Bacteriol. 184:5508-5512.

163. Braun, M., F. Endriss, H. Killmann, and V. Braun. 2003. In vivo

reconstitution of the FhuA transport protein of Escherichia coli K-12. J.

Bacteriol. 185:5508-5518.

164. Ghosh, J. and K. Postle. 2004. Evidence for dynamic clustering of

carboxy-terminal aromatic amino acids in TonB-dependent energy

transduction. Mol. Microbiol. 51:203-213.

165. Zwick, M. B., L. L. Bonnycastle, K. A. Noren, S. Venturini, E. Leong,

C. F. Barbas, III, C. J. Noren, and J. K. Scott. 1998. The maltose-

binding protein as a scaffold for monovalent display of peptides derived

from phage libraries. Anal. Biochem. 264:87-97.

166. Moeck, G. S. and L. Letellier. 2001. Characterization of in vitro

interactions between a truncated TonB protein from Escherichia coli and

the outer membrane receptors FhuA and FepA. J. Bacteriol. 183:2755-

2764.

167. Moeck, G. S., P. Tawa, H. Xiang, A. A. Ismail, J. L. Turnbull, and J.

W. Coulton. 1996. Ligand-induced conformational change in the

ferrichrome-iron receptor of Escherichia coli K-12. Mol. Microbiol.

22:459-471.

168. Mandava, S., L. Makowski, S. Devarapalli, J. Uzubell, and D. J. Rodi.

2004. RELIC--a bioinformatics server for combinatorial peptide analysis

and identification of protein-ligand interaction sites. Proteomics. 4:1439-

1460.

Page 232: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

208

169. Makowski, L. and A. Soares. 2003. Estimating the diversity of peptide

populations from limited sequence data. Bioinformatics. 19:483-489.

170. Delano W.L. 2008. The Pymol Molecular Graphics System, DeLano

Scientific LLC, Palo Alto, CA, USA.

171. Morris, G. M., D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R.

K. Belew, and A. J. Olson. 1998. Automated docking using a Lamarckian

genetic algorithm and an empirical binding free energy function. J.

Comput. Chem. 19:1639-1662.

172. Barnard, T. J., M. E. Watson, Jr., and M. A. McIntosh. 2001.

Mutations in the Escherichia coli receptor FepA reveal residues involved

in ligand binding and transport. Mol. Microbiol. 41:527-536.

173. Chakraborty, R., E. A. Lemke, Z. Cao, P. E. Klebba, and D. van der

Helm. 2003. Identification and mutational studies of conserved amino

acids in the outer membrane receptor protein, FepA, which affect transport

but not binding of ferric-enterobactin in Escherichia coli. Biometals

16:507-518.

174. Köster, W. and V. Braun. 1990. Iron (III) hydroxamate transport into

Escherichia coli. Substrate binding to the periplasmic FhuD protein. J.

Biol. Chem. 265:21407-21410.

175. Schultz-Hauser, G., W. Köster, H. Schwarz, and V. Braun. 1992.

Iron(III) hydroxamate transport in Escherichia coli K-12: FhuB-mediated

membrane association of the FhuC protein and negative complementation

of fhuC mutants. J. Bacteriol. 174:2305-2311.

176. Lee, Y. H., R. K. Deka, M. V. Norgard, J. D. Radolf, and C. A.

Hasemann. 1999. Treponema pallidum TroA is a periplasmic zinc-

binding protein with a helical backbone. Nat. Struct. Biol. 6:628-633.

177. Lawrence, M. C., P. A. Pilling, V. C. Epa, A. M. Berry, A. D.

Ogunniyi, and J. C. Paton. 1998. The crystal structure of pneumococcal

surface antigen PsaA reveals a metal-binding site and a novel structure for

a putative ABC-type binding protein. Structure. 6:1553-1561.

178. Krewulak, K. D., R. S. Peacock, and H. J. Vogel. 2004. Periplasmic

Binding Proteins Involved in Bacterial Iron Uptake, p. 113-129. In Iron

Transport in Bacteria. ASM Press, Washington DC.

179. Rohrback, M. R., S. Paul, and W. Köster. 1995. In vivo reconstitution of

an active siderophore transport system by a binding protein derivative

lacking a signal sequence. Mol. Gen. Genet. 248:33-42.

Page 233: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

209

180. Carter, D. M., J. N. Gagnon, M. Damlaj, S. Mandava, L. Makowski,

D. J. Rodi, P. D. Pawelek, and J. W. Coulton. 2006. Phage display

reveals multiple contact sites between FhuA, an outer membrane receptor

of Escherichia coli, and TonB. J. Mol. Biol. 357:236-251.

181. Yang, Y. R. and H. K. Schachman. 1996. A bifunctional fusion protein

containing the maltose-binding polypeptide and the catalytic chain of

aspartate transcarbamoylase: assembly, oligomers, and domains. Biophys.

Chem. 59:289-297.

182. Lebowitz, J., M. S. Lewis, and P. Schuck. 2002. Modern analytical

ultracentrifugation in protein science: a tutorial review. Protein Sci.

11:2067-2079.

183. Myszka, D. G. 1999. Improving biosensor analysis. J. Mol. Recognit.

12:279-284.

184. Lamb, A. L., A. S. Torres, T. V. O'Halloran, and A. C. Rosenzweig.

2000. Heterodimer formation between superoxide dismutase and its

copper chaperone. Biochemistry 39:14720-14727.

185. Mader, C., C. Huber, D. Moll, U. B. Sleytr, and M. Sára. 2004.

Interaction of the crystalline bacterial cell surface layer protein SbsB and

the secondary cell wall polymer of Geobacillus stearothermophilus PV72

assessed by real-time surface plasmon resonance biosensor technology. J.

Bacteriol. 186:1758-1768.

186. Nakajima, H., L. Cocquerel, N. Kiyokawa, J. Fujimoto, and S. Levy.

2005. Kinetics of HCV envelope proteins' interaction with CD81 large

extracellular loop. Biochem. Biophys. Res. Commun. 328:1091-1100.

187. Sprencel, C., Z. Cao, Z. Qi, D. C. Scott, M. A. Montague, N. Ivanoff, J.

Xu, K. M. Raymond, S. M. Newton, and P. E. Klebba. 2000. Binding of

ferric enterobactin by the Escherichia coli periplasmic protein FepB. J.

Bacteriol. 182:5359-5364.

188. Borths, E. L., B. Poolman, R. N. Hvorup, K. P. Locher, and D. C.

Rees. 2005. In vitro functional characterization of BtuCD-F, the

Escherichia coli ABC transporter for vitamin B12 uptake. Biochemistry

44:16301-16309.

189. Carter, D. M., I. R. Miousse, J. N. Gagnon, E. Martinez, A. Clements,

J. Lee, M. A. Hancock, H. Gagnon, P. D. Pawelek, and J. W. Coulton.

2006. Interactions between TonB from Escherichia coli and the

periplasmic protein FhuD. J. Biol. Chem. 281:35413-35424.

Page 234: Interactions between energy transducer TonB and …digitool.library.mcgill.ca/thesisfile86876.pdf · Interactions between energy transducer TonB and ferric hydroxamate transport proteins

210

190. Karlsson, R., P. S. Katsamba, H. Nordin, E. Pol, and D. G. Myszka.

2006. Analyzing a kinetic titration series using affinity biosensors. Anal.

Biochem. 349:136-147.

191. Morris, G. M., R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D.

S. Goodsell, and A. J. Olson. 2009. AutoDock4 and AutoDockTools4:

Automated docking with selective receptor flexibility. J. Comput. Chem.

Epub ahead of print

192. Pettersen, E. F., T. D. Goddard, C. C. Huang, G. S. Couch, D. M.

Greenblatt, E. C. Meng, and T. E. Ferrin. 2004. UCSF Chimera--a

visualization system for exploratory research and analysis. J. Comput.

Chem. 25:1605-1612.

193. Ködding, J. F. 2004. Biochemical and X-ray crystallographic studies on

the energy transducing protein TonB and the TonB-dependent siderophore

receptor FhuA from Escherichia coli. Ph.D. thesis. University of

Konstanz.

194. Dyson, H. J. and P. E. Wright. 2005. Intrinsically unstructured proteins

and their functions. Nat. Rev. Mol. Cell Biol. 6:197-208.

195. Blommel, P. G. and B. G. Fox. 2007. A combined approach to improving

large-scale production of tobacco etch virus protease. Protein Expr. Purif.

55:53-68.

196. Lapko, A., A. Müller, O. Heese, K. Ruckpaul, and U. Heinemann.

1997. Preparation and crystallization of a cross-linked complex of bovine

adrenodoxin and adrenodoxin reductase. Proteins 28:289-292.

197. Leach, M. R., J. W. Zhang, and D. B. Zamble. 2007. The role of

complex formation between the Escherichia coli hydrogenase accessory

factors HypB and SlyD. J. Biol. Chem. 282:16177-16186.