Biogenesis of [Fe-S] proteins in Escherichia coli

Frederic Barras, LCB, CNRS, Marseille

Marburg, 21 april 2004

Biogenesis of [Fe-S] proteins in Escherichia coli

Background

The IscS system: other’s story

The Suf system: our story

The Csd system: the new story

Some [Fe-S] clusters

Kiley and Beinert, 2003

Biological functions of [Fe-S] clusters

Electron transfer

Sulfur transfer

Sensing O2 and derivatives

[Fe-S] cluster containing proteinsFNR

PJ Kiley

[Fe-S] cluster containing proteinsAconitase/IRE-BP

Aconitase[4Fe-4S] cluster

IRE-binding site not accessible

IRE-BPNo [4Fe-4S] cluster

IRE-binding site accessible

[2Fe-2S]3+

[Fe-S] cluster containing proteinsSoxR

-35

-35

-10

-10

[2Fe-2S]2+ [2Fe-2S]2+

soxS mRNA

[2Fe-2S] 3+

Pomposiello and Demple, 2001

How to make [Fe-S] proteins?1- Chemist’s answer

Apo-protein [Fe-S] protein

Fe2+

Na2SDTT

How to make [Fe-S] proteins?2- Biologist’s answer

Source of Fe and S?(toxicity)

Assembly of [Fe-S] cluster(insertion and folding)

Biogenesis of [Fe-S] proteins in Escherichia coli

Background

The IscS system: other’s story

The Suf system: our story

The Csd system: the new story

Study of nitrogen fixation in Azotobacter vinelandiiDr. Dean’s laboratory (1993)

Apo-nitrogenase [4Fe-4S] nitrogenase

Fe2+

CysteineDTT

NifS

Enzymatic activity of Cysteine desulfurases

NifS-like gene clusters

Frazzon and Dean, 2003Muhlenhoff and Lill, 2000

A. vinelandii

A. Vinelandii(isc)

E. coli(isc)

iscR nifS iscU iscA hscB hscA fdx

R. prowazekii(isc)

hscB hscA fdxiscS1 iscS2 iscU iscA1 iscA2

NFS1 ISU1,2 ISA1,2 JAC1 SSQ1 YAH1 ARH1 S. cerevisiae

Functions of the isc gene products

iscR iscS iscU iscA hscB hscA fdx

[2Fe-2S]

H2O2

-Cysteine

desulfurase

[Fe-S] cluster assembly scafold

Molecular chaperones

[2Fe-2S] Ferredoxin

Model

IscS

IscU

IscSIscA

Fdx

Apo

[Fe-S]

Cysteine

AlanineS

[Fe-S]

Cysteine

Alanine

S[Fe-S]

HscA BATP

ADP

3 NifS-like in E. coli

iscS csdA (CSD) sufS (csdB)

location 53.7’ 37.8’ 63.4’

AminotransferaseClass V

I II II

SSGSACTS RXGHHCA

Structure 3D + +

Cysteine Selenocysteine

0.38 3.1

0.9 6.2

0.025.5

Loci containing NifS homologues in E. coli

iscR iscS iscU iscA

sufC sufS sufEsufA sufB sufD

csdA ygdK ygdL

Biogenesis of [Fe-S] proteins in Escherichia coli

Background

The IscS system: other’s story

The Suf system: our story

The Csd system: the new story

EnterobacteriaPlant pathogen

Search for virulence genes by transposon mutagenesis

Beaulieu and van Gijsegem, J Bact, 1990

Erwinia chrysanthemi

sufA sufB sufC sufD sufS sufE

pin10: suf genes RT-PCR C PCR

‚ ƒ

‚ ƒ

0

500

1000

1500

2000

0 200 400 600 800min

-gl

ucur

onid

ase

actv

ity wt - FeSO4

fur + FeSO4

fur - FeSO4

wt + FeSO4

Fur regulated operon

Patzer and Hantke, J Bact 1999, Nachin et al., Mol. Microbiol. 2001

sufA sufB sufC sufD sufS sufE

Functional Prediction

SignaturesABC ATPase

? ? ?

IscA

Cluster [Fe-S] formation

NifS-like

Cysteine desulfurase

SufBSufD

Takahashi et al., JBC, 2002

sufA sufB sufC sufD sufS sufEErwinia chrysanthemi

Archeoglobus fulgidusMethanococcus jannaschiiM. thermoautotrophicumPyrococcus abyssiPyrococcus horikoshii

Archaebacteria

Aquifex aeolicus

Chlamidiae pneumoniaeChlamidiae trachomatisDeinococcus radioduransEscherichia coliMycobacterium tuberculosisSynechocystis spp.Thermotoga maritimaTreponema pallidiumXylella fastidiosa

Bacillus subtilis

-

-

-

Eubacteria

sufB sufC sufD

ABC transporter ?

Walker A and B boxes C region

No TM No TM

SufC exhibits ATPase activity

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

0 0,5 1 1,5 2

[ATP] (mM)

M .

min

-1

Vm: 4.45 mole min-1

Km: 0.29 mM

Interactions SufB-SufC SufD-SufC

SufB

SufC

SufD

LexASuf

B

SufC

SufD

ActYeast Two-Hybrid

SufC (chromosomal) Ha-SufB

(plasmidic)SufC (plasmidic)

MsrA

OutF

Cel5

Total Peri CytoMbrs

Total Peri CytoMbrs

SufC and SufB are cytoplasmic

Total Peri CytoMbrs

Total Peri CytoMbrs

MsrA

OutF

Cel5

Suf C: an unorthodox cytoplasmic ABC ATPase

SufB

SufCSufD

ATP

ADP

Nachin et al., EMBO J. 2003

Fe-S cluster transfer from HoloSufA/IscA to apoBiotin Synthase.

holoSufA

holoIscA

(Fe2+ and S2-)

Mature BioB

X

Ollagnier de Choudens et al., JBC 2003

ApoBioB was incubated with 5 mM DTT and a two-fold molar excess of either holoSufA (X) or holoIscA () or a four-fold molar excess of Fe2+ and S2- () and increasing concentrations of bathophenantroline. After 30 minutes incubation at 18°C, biotin synthase activity was measured.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 50 100 150

nm

ol b

ioti

n/ n

mol

Bio

B m

onom

er

Bathophenantroline ( M)

Fe-S transfer « en bloc »

SufA

Apoprotein

[Fe-S]

Protein [Fe-S]

Fe-S

SufB

SufCSufD

ATP

ADP

Biochemical analysis

Vm (units/mg)

SufS 0.02

Csd 1.1

IscS 0.38

Units : µmol Ala / min

IscS from E. coli

Cupp-Vickery, JR et al. (2004) J. Mol. Biol. 330,1049.

Structural studies

Black: IscS from T. maritimaWhite: SufS from E. coli

Mihara, H. et al. (2002) J. Biochem. 131, 679-685

Cys324

Flexible loop

0

100

200

300

400

500

0 5 10 15 20 25 30 35 40

nm

ole

alan

ine/

nm

ol S

ufS

Time (min)

SufS is activated by SufE

SufS

SufS

SufS+SufE

SufS+SufE

Cysteine

Selenocysteine

Loiseau et al., JBC 2003

Biochemical analysis

Vm (units/mg)

SufS 0.021

SufS+SufE 0.750

SufS(C369S) 0.0006

SufS(C369S)+SufE 0.001

A

B

C

Sulfur transfer from SufS to SufE

Alanine

Cysteine

SufS-S364-SH

SufS-S364H

SufE-S51H

SufE-S51-SH DTT

« S2- »

H2N CH C

CH2

OH

O

SH

H2N CH C

CH3

OH

O

SufA

SufE

Apoprotein

[Fe-S]

Protein [Fe-S]

SufS

Cysteine

S2-

Fe-S

SufB

SufCSufD

ATP

ADP

Fe 2+ ???

Suf : a [Fe-S] insertion machinery

Fumarase

Glut Synthase

PGM

wt

126+/-16

128+/-2

37+/-6

+PMS

16+/-2

313+/-1

36+/-9

ratio

13%

244%

97%

sufC

138+/-13

124+/-4

35+/-10

+PMS

9+/-2

283+/-3

39+/-6

ratio

6%

228%

111%

Physiological role of Suf in E. coli

0

0,5

1

1,5

2

2,5

3

3,5

0 500 1000 1500

w t

delta suf

gnd

gnd delta suf

0

0,5

1

1,5

2

2,5

3

3,5

0 500 1000 1500

w t+

delta suf +

gnd +

gnd deltasuf +

MM + gluconateMM + glycerol gluconate

GND6PGDH(Fe-S)

Hypothesis

Suf protects oxygen labile [Fe-S] clusters

Iron acquisition in E. chrysanthemi:an essential virulence factor

Chr

ChrFe3+

Chr=chrysobactin

+Fe3+chryso +FeCl3Strains

cbs 19 1 20 1sufB cbs 12 1 20 1sufC cbs 0 20 1sufD cbs 0 20 1

Hypothesis:Suf is important for iron acquisition A basis for importance of Suf in virulence ?

Suf and iron acquisition in E. chrysanthemi

MM+dipyridyl

0 0 0 0

-

Biogenesis of [Fe-S] proteins in Escherichia coli

Background

The IscS system: other’s story

The Suf system: our story

The Csd system: the new story?

The new story

Csd ygdK ygdL

Cysteine desulfurase SufE-like ThiF-like

ThiF+ThiI+ThiJIscS

Thiamine biosynthesis early steps

ThiFATPO

OHThiS

O

OAMPThiS

O

SHThiS

Molecular analysiscomplex Csd/YgdK/YgdL

Csd

YgdK YgdL

Biochemical analysis

Vm (units/mg)

Csd 1.1

Csd+YgdK 2.5

Csd(C61)+YgdK ND

Biochemical analysisSulfur transfer

Csd

YgdK

YgdL

Cysteine

AlanineS

S

S

Targets

YgdLactive

?

?

Perspectives

•Iron source ?

•Mechanistic aspectsRole of SufBCD ?Role of HscAB ?Role of Fdx ?Role of IscA vs IscU?…………

•Co-translational insertion vs post-translational repair

Perspectives

Substrate and/or Environmental specificity of each system ?

Isc

Enz-SSH Thiamin

CsdSuf

[Fe-S]O

ThiSIscS-S-S

Fe

Fe

Fe

Fe

S

S

SS

[Fe-S]

[Fe-S] Protein Oxidized [Fe-S]

*

Apoprotein

ROS

Isc Suf

Genetic interaction between Isc and Suf

Redundancy?

Synthetic lethality

iscS - sufABCSDE

Suppression

iscS - psufABCSDE

Takahashi et al., JBC, 2002

Outen et al., Mol Mic, 2004

[Fe-S]

[Fe-S] Protein Oxidized [Fe-S]

*

Apoprotein

ROS

Isc Suf

GeneticSuppression

iscRSUA NAD-

iscRSUA

/pcsdAygdK

NAD+

Redundancy?

Imminoaspartate + DHAP

Quinolinic acid

NAD

NadA [4Fe-4S]

[Fe-S]

[Fe-S] Protein Oxidized [Fe-S]

*

Apoprotein

ROS

Isc SufCsd

Strains Growth under iron limitation

(Dipyridyl 0.32mM)

sufA +sufB -sufC -sufC + psufCK40A -sufD -sufS -sufE -sufE + psufEC51S -

Suf required under iron limitation

Strains Growth under iron limitation

iscS +

sufS + piscS -

csdA +

ygdK +

sufS + pcsdA-ygdK

-

Suf is specifically required under iron limitation

Isc

Csd

Suf

Fe-SThionucleosides

Iron assimilation

ThiaminMolybdopterin

Substrate specificity

Isc

Csd

Suf

H2O2 OxyR

Environmental specificity

IscR[2Fe-2S]

Fur

Nachin et al., 2001,, Schwartz et al., 2001, Zheng et al., 2001, Hantke, 2002 Lee et al., 2004

Actors

LCB, MarseilleLaurent LoiseauLaurence Nachin

CEA, GrenobleSandrine Ollagnier de Choudens

Marc Fontecave

INAPG, ParisDominique Expert

Collaborators