Investigations into the Investigations into the Protein-Protein Interactions and Protein-Protein Interactions and Mechanism of Inhibition for the Mechanism of Inhibition for the

ADP-Ribosyl Transferase Reaction of ADP-Ribosyl Transferase Reaction of Pseudomonas aeruginosaPseudomonas aeruginosa Exotoxin A Exotoxin A

Susan P. Yates

CHEM 795

OutlineOutline

• Background– Pseudomonas aeruginosa

– Exotoxin A (ETA)

– Eukaryotic elongation factor –2 (eEF-2)

• Objectives– Characterization of a loop region

– ETA-eEF-2 interactions

– Organic-based inhibitors

• Conclusions

Pseudomonas aeruginosaPseudomonas aeruginosa• Gram negative rod• Found in soil and water

– adapts to new environments • hospitals

• Opportunistic pathogen– infects when host defenses are

impaired• AIDS, cancer, severe burns,

cystic fibrosis

• Several virulence factors– cell associated or secreted factors

• pili, adhesions, LPS, Exotoxin A, exoenzyme S, alginate

– antibiotic resistance

(Dennis Kunkel/Dennis Kunkel Microscopy, 2001)

Exotoxin AExotoxin A

• Member of the mono-ADP-ribosyl transferase (ADPRT) family

• Secreted factor of P. aeruginosa– 66 kDa single polypeptide

– three functional domains

– proenzyme that is activated within the eukaryotic cell

• proteolytic event

– Most potent virulence factor

(Allured et al (1986) PNAS, 83:1320)

Domain I: Domain I: Receptor-Binding Receptor-Binding

Domain II: Domain II: TranslocationTranslocation

Domain III: Domain III: CatalyticCatalytic

ADP-Ribosylation of eEF-2 by ETAADP-Ribosylation of eEF-2 by ETA

ADP-ribosyl-eEF-2ADP-ribosyl-eEF-2

nicotinamidenicotinamide

N

O

NH2

OHOH

OHOH NH2

O

N

N N

NCH2

P O-O

O

O

O-O P

OCH2 O

+CH3

CH3

CH3O

H2NN

PROTEINCH2

N N

H

NADNAD++

eEF-2eEF-2

O

NH2

+

O

NCH2

O

P-O O

O

O

-O OP

CH2 O

N

N N

N

NH2OH

OH

OHOH

N N

CH2 PROTEIN

NH2N

O CH3

CH3

CH3+

:

H

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

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

-TAD-TAD

HisHis440440

GluGlu553553

TyrTyr481481

TyrTyr470470

Eukaryotic Elongation Factor 2 (eEF-2)Eukaryotic Elongation Factor 2 (eEF-2)

• 90 –110 kDa protein• Protein substrate for ETA• Important factor in the

elongation step of protein synthesis

• Covalent modification by ETA produces ADP-ribosyl eEF-2– prevents its participation

in translation

– protein synthesis ceases• cell death

(Gomez-Lorenzo et al (2000) EMBO J 19:2710)

tRNAtRNA

eEF-2eEF-2

ObjectivesObjectives

• Characterize the role of a loop region in ETA– catalytic or eEF-2-substrate binding loop

• Toxin-eEF-2 interactions– pH profile– mapping the eEF-2 binding site on PE24 using

fluorescence

• Inhibition– NAP derivatives– PARP inhibitors

ObjectivesObjectives

• Characterize the role of a loop region in ETA– catalytic or eEF-2-substrate binding loop

• Toxin-eEF-2 interactions– pH profile– mapping the eEF-2 binding site on PE24 using

fluorescence

• Inhibition– NAP derivatives– PARP inhibitors

ObjectivesObjectives

• Characterize the role of a loop region in ETA– catalytic or eEF-2-substrate binding loop

• Toxin-eEF-2 interactions– pH profile– mapping the eEF-2 binding site on PE24 using

fluorescence

• Inhibition– NAP derivatives– PARP inhibitors

Characterization of a Loop in ETACharacterization of a Loop in ETA

• History of Loop C– functional removal

diminishes activity 1.8 x 10-4-fold

– retains wild-type NAD+ binding

• Alanine-scanning mutagenesis– some muteins exhibited

reduced activity

– dissociation and Michaelis constants for NAD+ similar to wild-type

• What is the role of Loop C?– catalytic? or

– eEF-2 substrate binding?

-TAD-TAD

Loop CLoop C

ADP-Ribosylation Activity of Loop C ADP-Ribosylation Activity of Loop C Determination of KDetermination of Kmm for eEF-2 for eEF-2

(Yates & Merrill (2001) JBC 276:35029 )

• pG-Loop C– each residue replaced with glycine

– functional removal of Loop C

– activity too low to determine Km(eEF-2)

Can Loop C Bind eEF-2?Can Loop C Bind eEF-2?

• Fluorescence resonance energy transfer (FRET) eEF-2 binding assay– no Cys in wild-type PE24

• introduce single Cys

• label with IAEDANS

– label eEF-2 with fluorescein

– perform FRET• titrate with fluorescein labeled eEF-2

Using FRET to Study eEF-2-Binding to ETAUsing FRET to Study eEF-2-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

• absorbance spectrum of acceptor overlaps fluorescence emission spectrum of donor

• dipole-dipole interactions are parallel

PE24-AEDANS

eEF-2-Fluorescein

Labeling Toxin with IAEDANS (Donor)Labeling Toxin with IAEDANS (Donor)

S CH2 PROTEIN

O

CH2CNHCH2CH2NH

SO3HSO3H

NHCH2CH2NH C CH2 I

O

PROTEINCH2HS..

Protein adduct

IAEDANS

Labeling eEF-2 with Fluorescein (Acceptor)Labeling eEF-2 with Fluorescein (Acceptor)

Protein adduct

OHO O

C OH

O

NH C CH2 I

..HS CH2 PROTEIN

PROTEINCH2 SCH2CNH

O

OHC

OHO O

Fluorescein

eEF-Binding of pG-Loop CeEF-Binding of pG-Loop C

0 1000 2000 3000 4000 5000

0.0

0.2

0.4

0.6

0.8

1.0

S585C-AEDANS pG-Loop C/S585C-AEDANS

Fra

ctio

nal

Sat

ura

tio

n (F

/F

max

)

[eEF-2--5AF], (nM)

• Created mutein pG-Loop C with S585C– labeled Cys at 585 with IAEDANS

• Dissociation constants (KD)– S585C-AEDANS

• 1471 ± 76 nM

– pG-Loop C/S585C-AEDANS

• 1526 ± 76 nM

(Yates & Merrill (2001) JBC 276:35029 )

Loop C FunctionLoop C Function

• Modulates transferase activity – catalytic element

• Stabilization of the transition state structure within the active site – alignment

– favourable interactions

• Q483, D484, Q485 (blue)

-TAD-TADTyrTyr481481

TyrTyr470470

(Yates & Merrill (2001) JBC 276:35029 )

ObjectivesObjectives

• Characterize the role of a loop region in ETA– catalytic or eEF-2-substrate binding loop

• Toxin-eEF-2 interactions– pH profile– mapping the eEF-2 binding site on PE24 using

fluorescence

• Inhibition– NAP derivatives– PARP inhibitors

Effect of pH on eEF-2 Binding to ETAEffect of pH on eEF-2 Binding to ETA• Utilizes the FRET

eEF-2 binding assay• Optimum eEF-2

binding at pH 7.8

• Two distinct pKa values – acidic pKa ~ 6.3

• His residue

• His 440?

– alkaline pKa ~ 9.3

• Tyr residue

• Tyr 481?

pH Profiles of eEF-2 Binding and CatalysispH Profiles of eEF-2 Binding and Catalysis

Catalysis eEF-2 Binding

(Armstrong & Merrill (2001) Anal Biochem 292:26 )

• eEF-2 binding may be responsible for pH dependence in catalysis

Mapping the eEF-2 Binding Site-A Mapping the eEF-2 Binding Site-A Fluorescence Quenching ApproachFluorescence Quenching Approach

• series of surface-exposed single cysteine mutants of PE24 were labeled with the fluorophore, IAEDANS

Front ViewFront View

S44S4499T442T442

S459S459

S515S515

S507S507

S585S585

S408S408

S410S410

R490R490

E486E486Back ViewBack View R490R490

E486E486

S44S4499 T442T442

S459S459

S515S515S507S507

S585S585

S408S408

S410S410

• Monitor the fluorescence change of these protein adducts upon eEF-2 binding – contact regions show fluorescence quenching in the

presence of unlabeled eEF-2

– regions that do not contact show little fluorescence change upon addition of eEF-2

Mapping the eEF-2 Binding Site-A Mapping the eEF-2 Binding Site-A Fluorescence Quenching ApproachFluorescence Quenching Approach

FRET versus Fluorescence FRET versus Fluorescence QuenchingQuenching

• FRET– requires a

donor/acceptor pair

– donor and acceptor pair within 10-100 Å of each other

– measures global interactions

• Fluorescence quenching– no acceptor, only one

component contains the fluorophore

– quenching requires direct interaction

– measures local interactions

eEF-2 Binding to PE24-AEDANSeEF-2 Binding to PE24-AEDANS

0 1000 2000 3000 4000 5000 6000

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

nal

Sat

ura

tio

n (F

/F

max

)

[eEF-2], nM

S585C-AEDANS S507C-AEDANS S449C-AEDANS

Environment of IAEDANS ProbeEnvironment of IAEDANS Probe

• IAEDANS probe is sensitive to its chemical environment– partly reflected by observed

spectral shifts(Hudson & Weber (1973) Biochemistry 12:4154)

Preliminary Map of Sites of eEF-2 Preliminary Map of Sites of eEF-2 AssociationsAssociations

• eEF-2 contacts a large area of the toxin – near active site and upper

portion in structure

– contact minimal at base of structure

• S585C-AEDANS

• S507C-AEDANS

– current data cannot distinguish direct protein-protein interaction from conformational change

S507C-AEDANSS507C-AEDANS

S585C-AEDANSS585C-AEDANS

Understanding Toxin-eEF-2 interactionsUnderstanding Toxin-eEF-2 interactions• Future work

– higher resolution map of sites on PE24 that associate with eEF-2

• introduce new Cys residues – near 585 and 507 to confirm

absence of contact

– explore new regions

– acrylamide quenching• accessibility of IAEDANS probe

• contact versus conformational change

– measure intermolecular distances using FRET at each site on toxin to eEF-2

S507C-AEDANSS507C-AEDANS

S585C-AEDANSS585C-AEDANS

ObjectivesObjectives

• Characterize the role of a loop region in ETA– catalytic or eEF-2-substrate binding loop

• Toxin-eEF-2 interactions– pH profile– mapping the eEF-2 binding site on PE24 using

fluorescence

• Inhibition– NAP derivatives– PARP inhibitors

Inhibition of ETA Activity-The History of NAPInhibition of ETA Activity-The History of NAP

• 1,8 naphthalimide (NAP) is a potent inhibitor of toxin activity– IC50 of 87 ± 12 nM

– competitive inhibitor with Ki of 45 ± 5 nM

– NAD+ dissociation constant of 56 ± 6 nM

– reversible inhibition• non-covalent association

– molecular modeling suggests it binds in nicotinamide binding pocket

– transition-state analog

N

H

OO

• PROBLEM– very low water solubility

NAPNAP

NAP DerivativesNAP Derivatives• Create a library of NAP

derivatives– maintain or enhance

inhibitory properties of NAP

– goal is to increase water solubility

• in vitro testing– IC50

– mechanism of inhibition

– inhibitory constant, Ki

– NAD+ binding constant

N

R

OO

R1

N

R

OO

R1

R2

N

R

OO

R1

R2

XH

GN

R

O

R1, R2 = alkyl groups, halogen, carboxylic acid derivatives, aldehydes X = oxygen, nitrogen G = S=O, SO2, P(O)H

Alternatives to the NAP Family-Alternatives to the NAP Family-PARP InhibitorsPARP Inhibitors

• Poly(ADP-ribose) polymerases (PARPs)– DNA repair enzyme

• Involved in chromatin decondensation, DNA replication and repair, gene expression, malignant transformation, cellular differentiation and apoptosis

– poly-ADP ribosylation on target protein– catalytically and structurally related to ETA

5-AIQ – A PARP Inhibitor5-AIQ – A PARP Inhibitor

NH

O

NH3+Cl

-

O

NH2

N nicotinamidenicotinamide

• 5-aminoisoquinolinone (5-AIQ)– mimic of the nicotinamide moiety of NAD+

– water soluble

– IC50 of 240 nM in vitro for PARP enzyme (Suto et al, 1991)

– human cardiac myoblasts showed concentration-dependent inhibition of PARP activity (IC50 ~12 M) (McDonald et al, 2000)

5-AIQ5-AIQ

Inhibition of ETA by 5-AIQInhibition of ETA by 5-AIQ

• IC50 139 ± 15 M (Armstrong (2001) PhD thesis) 0 20 40 60 80 100

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Rea

ctio

n R

ate

(M

/sec

)

[5-AIQ], M

NH

O

NH3+Cl

-

O

NH2

N

• IC50 23 ± 3 M

nicotinamidenicotinamide

5-AIQ5-AIQ

ConclusionsConclusions• Loop C within ETA serves a catalytic role

– stabilization of the transition state

• eEF-2 binding is pH-dependent– His and Tyr implicated

– eEF-2 binding may be responsible for the observed catalytic pH dependence

• Regions of association on ETA– eEF-2 may bind near the active site with little contact near

base of structure• conformational changes another possibility

• 5-AIQ improved level of inhibition compared to nicotinamide – not as potent inhibitor as NAP to warrant further study

AcknowledgementsAcknowledgements• Supervisor

– Dr. A. R. Merrill

• Advisory Committee– Dr. R. Keates– Dr. J. Lam– Dr. J. Honek

• Organic synthesis– Dr. A. Schwan– Mike Ganton

• Merrill Lab– Tanya Brodeur– Abdi Musse– Janine Passi– Gerry Prentice– Tania Roberts– Dave Teal

– Paula Russell

• Funding Agencies– CIHR

– CCFF