Shining Light on the ADP-Shining Light on the ADP-Ribosylation Mechanism Ribosylation Mechanism of Pseudomonas of Pseudomonas ToxinToxin

A.R. Merrill

Dept. of Chem. & Biochem.

Univ. of Guelph

2

Guelph, Ontario, Canada

3

P. aeruginosa ubiquitous, Gram-neg

bacterium--Pathogenic cystic fibrosis, cancer, burn,

AIDS, and post-operative patients

infections: acute localized to systemic

leukopenia, circulatory collapse, liver, kidney, and skin necrosis, hemorrhaging, corneal destruction, and pneumonia

most virulent factor--Exotoxin A(Dennis Kunkel; Microscopy, 2001)

4

Exotoxin A

66 kDa protein secreted by P. aeruginosaLD50 = 0.2 g/kg (mice)

mono-ADPRT enzyme • related to diphtheria, cholera, tetanus,

pertussis toxins, PARPscellular effect: inhibition of protein

synthesis by alteration of elongation factor 2 (eEF2)

5

Intoxication Mechanism

H+

Intact ETA

28 kDa fragment

37 kDa fragment

ETA Receptor

Furin-like enzyme

Disulfide bond

6

Crystal Structure of ETA

Domain Ia

Domain Ib

Domain II

Domain III

(Wendekind et al., JMB 2001)

7

ADPRT Reaction

+

:N N

CH2 CH NH C

O

CH2 CH2 CH C O NH2

N(CH3)3 O

N

NH2

O

OH HO

CH2 O P O P O O

O-

O

O- H2C O

OH HO

O

N

N N

N

H2N

+

+

N

NH2

O

O

OH HO

CH2 O P O P O O

O-

O

O- H2C O

OH HO

O

N

N N

N

H2N

N N

CH2 CH NH C

O

CH2 CH2 CH C O NH2

N(CH3)3 +

eEF2 diphthamide residueNAD+

nicotinamide

ADP-ribosylated eEF2 diphthamide residue

+ H+

8

Catalytic Domain of ETA (PE24)Catalytic Domain of ETA (PE24)

(Li et al (1996) PNAS 93:6902)

-TAD-TAD

HisHis440440

GluGlu553553

TyrTyr481481

TyrTyr470470

9

Eukaryotic Elongation Factor 2 eEF2 is a soluble 94-97 kDa Forms binary complexes

with guanine nucleotides Complex formation

conformation change in eEF2 bind with high affinity to ribosomes

eEF2 catalyzes the translocation of peptidyl-tRNA on the ribosome in protein elongation

Gomez-Lorenzo et al., 2000

17.5Å EM Structure

tRNA

10

Structure of Yeast eEF2

(Jorgensen et al., Nat. Struct. Biol. 10, 387-385, 2003)

DiphthamideIV

11

Regulation of eEF2

A diphthamide residue is the site of regulatory modification on eEF2

eEF2 a substrate for cellular ADPRTs which function to regulate protein synthesis as part of normal metabolism

O

NH

NH

N+N(CH3)3

H2N

O

diphthamide

Bacterial toxins exploit the existing system

12

ADPRT Reaction

k 1

k -1

k 2

k -2

k 3

k -3E + NAD

+ E-NAD

+ E-NAD

+~eEF2 E-Nic~eEF2~ADPR

E-Nic eEF 2~ADPR

k 4 k -4 k 5 k -5

E E-NAD+ E-NAD+~eEF2 E-Nic~eEF2~ADPR E-Nic E

NAD+ eEF2 eEF2~ADPR Nic + H+

13

Principles of Fluorescence

S1

S0

S2

EXCITATION EMISSION

Absorption Fluorescence

14

Protein Fluorescence

(Lakowicz, 1983)

Trp

Tyr

Phe

15

Tryptophan Fluorescence

The intrinsic probe of choice for protein studies:• absorption and emission distribution extend further• strongest absorbance• large fluorescence intensity• most sensitive to local environment

Sensitivity due to 10 electrons of indole ring• 1La and 1Lb transitions

• dipole

16

ETA Kinetic Parameters

Substrate

Parameter NAD+ eEF2

KM (M) 275 52 8.0 1.8

Vmax (pmol.min-1) 234 30 258 24

kcat (min-1) 675 85 734 67

kcat/KM (M-1.min-1) 2.5 106 92.8 106

Armstrong & Merrill (2001) Anal. Biochem.292, 26-33.

17

Steady State Fluorescence

Change in fluorescence intensity as a function of substrate/ligand concentration

Fi/Fmax

[ligand]

Fi

[ligand]

1.0 -

18

NAD Binding

0 10 20 30 40 50 60 70 80 90200

300

400

500

600

0.005 0.010 0.015 0.020

1.85

1.90

1.95

2.00

2.05

2.10

2.15

2.20

2.25

(F

/Fm

ax)

B.

[NAD+] M

Fra

cti

on

al S

atu

rati

on

0 200 400 600 800 1000

0.0

0.2

0.4

0.6

0.8

1.0

1.2

A.

k ob

s (s-1

)

Time (seconds)

Flu

ore

scen

ce (volt

s)

Armstrong & Merrill, Biochemistry, in press

Kd=50 M

19

Stopped Flow Fluorescence

Kinetic data fit to exponentials:• Single exponential

• Multiple exponential)e(1FFF kt

10

...)e(1F)e(1FFF tk2

tk10

21

20

Kinetic and Thermodynamic Parameters for ADPRT Substrates

Table: Kinetic and Thermodynamic Parameters

for ADPRT Substrates and NAP Inhibitor of ETA

Parameter NAD+ eEF2 NAP

kon (M-1s-1)

4.7 0.4

320 39

82 ± 9

koff (s-1) 194 ± 15 131 ± 22 51 ± 6

koff/kon (M) 41 ± 3 0.41 ± 0.10 0.62 ± 0.07

Kd (M) 45 ± 5 0.71 ± 0.21 0.054 ± 0.006

1.2 ± 0.1

21

Ala Scan of Loop C (483-490)

*

483 484 485 486 487 488 489 4900.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e k

cat

Residue Number

Gln

Gln

AspAsp

Glu

Pro

AlaArg

Yates & Merrill (2001)

JBC 276,35029* *

**

22

eEF2 Docking Site

PE24

Loop C

Preparation of AEDANS--PE24 Adduct

NH N

H

O

I

HH

SO3

NH

SH

O

H

IAEDANS Cys residue of ETA mutant

NH N

H

O

SO3

S

H

N

O

H

AEDANS-labeled ETA mutant

+ HI

24

Toxin:eEF2 Interaction Models Identification of the contact sites between eEF2 and the

catalytic domain of ETA (PE24)• currently, this protein-protein interaction is poorly characterized

Two extreme models are possible• Minimal Contact Model – Maximum Contact Model

PE24eEF2

PE24eE

F2

25

Experimental Approach Single cysteine residues introduced into PE24 at

21 defined sites and labeled with the fluorophore, IAEDANS

• fluorescence studies performed in the presence and absence of eEF2

– acrylamide quenching

– fluorescence lifetime

– wavelength emission maximum

SO3H

NHCH 2CH2NH C CH2 I

O

G-525 A-519

S-515

Q-603 G-549

S-507

T-554

T-564

S-459

Q-415

S-408

Q-428

Q-592

S-410

R-490

N-577

S-449

T-442

E-486

S-585

A-476

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Emission Max and Lifetime4

08

41

04

15

42

84

42

44

94

59

47

64

86

49

05

07

51

55

19

52

55

49

55

45

64

57

75

85

59

26

03

-1

0

1

2

3

e

mm

ax

Residue Number

408

410

415

428

442

449

459

476

486

490

507

515

519

525

549

554

564

577

585

592

603

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

(

lifet

ime)

ns

Residue Number

No large shifts in emission maxima after eEF2 added• 3 nm red shift for S449C-

AEDANS and S515C-AEDANS• 1 nm blue shift for A519C-

AEDANS

No large changes in fluorescence lifetime after eEF2 added– Q428C-AEDANS (-1.2 ns)– A519C-AEDANS (1.2 ns)

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Fluorescence Lifetime Measurements

= 1/(kF + knF)

I(t) = I0e(-t/for a single exponential (one lifetime component)

I(t) =1e(-t/1) + 2e(-t/

2) + …

for multiple exponentials (multiple lifetime components)

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Acrylamide Quenching

Measure the ability of acrylamide to quench the fluorescence of AEDANS probe

– determine the bimolecular quenching constant (kq) in the presence and absence of eEF2

» Stern-Volmer equation

Protein

kq x 109M-1s-1 (– eEF2)

kq x 109M-1s-1 (+ eEF2)

1 S408C 1.03 0.02 0.37 0.01

2 S410C 1.29 0.01 0.36 0.01

3 Q415C 1.28 0.04 0.73 0.01

4 Q428C 0.86 0.03 0.54 0.01

5 T442C 0.56 0.02 0.20 0.01

6 S449C 0.60 0.02 0.27 0.01

7 S459C 0.94 0.03 0.42 0.02

8 A476C 0.89 0.03 0.58 0.01

9 E486C 1.29 0.04 0.48 0.02

10 R490C 1.27 0.04 0.70 0.02

11 S507C 0.80 0.02 0.33 0.01

12 S515C 0.55 0.03 0.41 0.01

13 A519C 1.23 0.04 0.61 0.02

14 G525C 0.96 0.01 0.75 0.02

15 G549C 0.65 0.03 0.35 0.01

16 T554C 0.88 0.04 0.32 0.01

17 T564C 0.50 0.02 0.27 0.01

18 N577C 1.10 0.04 0.74 0.01

19 S585C 0.90 0.02 0.59 0.01

20 Q592C 1.42 0.02 0.79 0.01

21 Q603C 0.68 0.02 0.39 0.01

1][0 QKF

FSV

i

qSV kK 0

F0/Fi

[Q]

KSV

1.0

29

Acrylamide Quenching

408

410

415

428

442

449

459

476

486

490

507

515

519

525

549

554

564

577

585

592

603

1.0

1.5

2.0

2.5

3.0

3.5

4.0

*

*

*

*

**

*

*

*

k q(-e

EF

2) /

kq(

+eE

F2)

Residue Number

Protein Adduct

kq(+ eEF 2) / kq(– eEF2)

S410C 3.53 S408C 2.79 T442C 2.77 T554C 2.74 E486C 2.69 S507C 2.44 S459C 2.26 S449C 2.25 A519C 2.04 T564C 1.85 R490C 1.82 G549C 1.79 Q592C 1.79 Q415C 1.75 Q603C 1.75 Q428C 1.60 A476C 1.54 S585C 1.54 N577C 1.48 S515C 1.34 G525C 1.28

30

Model of PE24-eEF2 Complex

Jørgensen, et al., (2003) Nat. Struc. Biol. 10, 379-385 (eEF-2 structure); Li et al (1996) PNAS 93, 6902 (PE24 structure)

Potential eEF2 contact sites on PE24 are shown as green spacefilled structures• minimal contact between

proteins

• diphthamide residue on eEF2 positioned near scissile glycosidic bond of NAD+ in active site

• two negative electrostatic patches on toxin and two positive electrostatic patches on eEF2 are aligned

a b

cd

1

2

3

4

5 6

7

8

9 PE24

eEF2

Domain IV

Diphthamide

31

FRET Experiments

R=Ro(E-1-1)1/6

E=1-FDA/FD

Ro=(K2JDAQDn-4)1/6 (9.79x103) Å

32

Using FRET to Study eEF2 Binding to ETAUsing FRET to Study eEF2 Binding to ETA Fluorescence Resonance

Energy Transfer (FRET)• transfer of excited state

energy from a donor to an acceptor

– no emission of a photon

• Criteria– donor and acceptor must be

in close proximity (10 – 100 Å)

– absorbance spectrum of acceptor overlaps fluorescence emission spectrum of donor

– dipole-dipole interactions are parallel

PE24-AEDANS

eEF2-Fluorescein

33

Labeling eEF2 with Fluorescein (Acceptor)Labeling eEF2 with Fluorescein (Acceptor)

Protein adduct

OHO O

C OH

O

NH C CH 2 I

..HS CH 2 eEF2

eEF2CH2 SCH 2CNH

O

OHC

OHO O

Fluorescein

34

PE24-AEDANS Binding with eEF2-PE24-AEDANS Binding with eEF2-5AF5AF

0 1000 2000 3000 4000

0.0

0.2

0.4

0.6

0.8

1.0

Fra

cti

on

al S

atu

rati

on

(F/F

max)

[eEF2-5AF], (nM)

Created S585C mutant toxin (WT activity)• labeled Cys at 585 with IAEDANS

Dissociation constant (Kd)– S585C-AEDANS

• 0.71 ± 0.08 M

(Armstrong et al. (2002) JBC 277:46669)

35

FRET Approach

T812C

T574C

PE24eEF2

Diphthamide

36

Future Work

Determine kinetic mechanism for mono- ADPRTs• Study movement of Loop C during catalysis

Develop inhibitors of ETA (competitive)• Crystallize PE24:inhibitor complexes

Characterize the nature of protein—protein interaction between ETA and eEF2• FRET Lifetime Analysis• Crystallize eEF2/TAD+/PE24 complex

Acknowledgments

Gerry Prentice Monica Tory Bryan Beattie Dr. Souzan Armstrong Susan Yates Dave Teal Patricia Taylor Dr. Jon Lamarre (U of Guelph) Dr. Art Szabo (WLU) Dr. David FitzGerald (NIH) Dr. Victor Marquez (NIH) Dr. Gilles Lajoie (UWO)

Funding

CIHR CCFF NSERC

38