1. Investigations into the Protein-Protein Interactions and Mechanism of Inhibition for the ADP-Ribosyl Transferase Reaction of Pseudomonas aeruginosa Exotoxin A Susan P. Yates CHEM 795

2. Outline 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

3. Pseudomonas 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)

4. Exotoxin 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: Receptor-Binding Domain II: Translocation Domain III: Catalytic

5. ADP-Ribosylation of eEF-2 by ETA

6. Catalytic Domain of ETA (PE24) (Li et al (1996) PNAS 93:6902)  -TAD His 440 Glu 553 Tyr 481 Tyr 470

7. 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) tRNA eEF-2

8. Objectives 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

9. Objectives 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

10. Objectives 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

11. Characterization 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 Loop C

12. ADP-Ribosylation Activity of Loop C Determination of K m 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 K m(eEF-2)

13. 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

14. Using 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

15. Labeling Toxin with IAEDANS (Donor) SCH 2 PROTEIN O CH 2 C NHCH 2 CH 2 NH SO 3 H 3 H NHCH 2 CH 2 NH CCH 2 I O PROTEINCH 2 HS.. Protein adduct IAEDANS  

16. Labeling eEF-2 with Fluorescein (Acceptor) Protein adduct O HO O COH O NH C CH 2 I.. HS CH 2 PROTEIN CH 2 S 2 C NH O OHC O HO O Fluorescein 

17. eEF-Binding of pG-Loop C Created mutein pG-Loop C with S585C –labeled Cys at 585 with IAEDANS Dissociation constants (K D ) –S585C-AEDANS 1471 ± 76 nM –pG-Loop C/S585C-AEDANS 1526 ± 76 nM (Yates & Merrill (2001) JBC 276:35029 )

18. Loop 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 Tyr 481 Tyr 470 (Yates & Merrill (2001) JBC 276:35029 )

19. Objectives 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

20. Effect 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 pK a values –acidic pK a ~ 6.3 His residue His 440? –alkaline pK a ~ 9.3 Tyr residue Tyr 481?

21. pH Profiles of eEF-2 Binding and Catalysis CatalysiseEF-2 Binding (Armstrong & Merrill (2001) Anal Biochem 292:26 ) eEF-2 binding may be responsible for pH dependence in catalysis

22. Mapping the eEF-2 Binding Site-A Fluorescence Quenching Approach series of surface-exposed single cysteine mutants of PE24 were labeled with the fluorophore, IAEDANS Front View S449 T442 S459 S515 S507 S585 S408 S410R490E486 Back View R490E486 S449 T442 S459 S515 S507 S585 S408 S410

23. 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 Fluorescence Quenching Approach

24. FRET versus Fluorescence Quenching 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

25. eEF-2 Binding to PE24-AEDANS

26. Environment of IAEDANS Probe IAEDANS probe is sensitive to its chemical environment –partly reflected by observed spectral shifts (Hudson & Weber (1973) Biochemistry 12:4154)

27. Preliminary Map of Sites of eEF-2 Associations 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-AEDANS S585C-AEDANS

28. Understanding 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-AEDANS S585C-AEDANS

29. Objectives 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

30. Inhibition of ETA Activity-The History of NAP 1,8 naphthalimide (NAP) is a potent inhibitor of toxin activity –IC 50 of 87 ± 12 nM –competitive inhibitor with K i 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 O O PROBLEM –very low water solubility NAP

31. NAP Derivatives Create a library of NAP derivatives –maintain or enhance inhibitory properties of NAP –goal is to increase water solubility in vitro testing –IC 50 –mechanism of inhibition –inhibitory constant, K i –NAD + binding constant N R O O R 1 N R O O R 1 R 2 N R O O R 1 R 2 XH G N R O R 1, R 2 = alkyl groups, halogen, carboxylic acid derivatives, aldehydes X = oxygen, nitrogen G = S=O, SO 2, P(O)H

32. Alternatives to the NAP Family- PARP 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

33. 5-AIQ – A PARP Inhibitor NH O NH 3 + Cl - O NH 2 N nicotinamide 5-aminoisoquinolinone (5-AIQ) –mimic of the nicotinamide moiety of NAD + –water soluble –IC 50 of 240 nM in vitro for PARP enzyme (Suto et al, 1991) –human cardiac myoblasts showed concentration- dependent inhibition of PARP activity (IC 50 ~12  M) (McDonald et al, 2000) 5-AIQ

34. Inhibition of ETA by 5-AIQ IC 50 139 ± 15  M (Armstrong (2001) PhD thesis) NH O NH 3 + Cl - O NH 2 N IC 50 23 ± 3  M nicotinamide 5-AIQ

35. Conclusions 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

36. Acknowledgements 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