1. Susan P. Yates Ph.D. Thesis Defence Supervisor: Dr. A. Rod Merrill
Protein-Protein Interactions and Inhibition of the ADP-Ribosyl Transferase Reaction of Pseudomonas aeruginosa Exotoxin A
Susan P. Yates
Ph.D. Thesis Defence
Supervisor: Dr. A. Rod Merrill

2. Outline Background Research Objectives Final Thoughts
Inhibition of the catalytic domain of exotoxin A
Interactions between the toxin and its protein substrate
Final Thoughts

3. Pseudomonas aeruginosa
Gram-negative rod-shaped bacterium
Opportunistic pathogen
Exploits some break in the host defenses to initiate an infection
Cystic fibrosis, severe burns, AIDS, cancer, etc.
Highly adaptable to new environments
Resistant to many antibiotics
Possesses a vast array of virulence factors
Very complex pathogenesis

4. Virulence Factors Pilus Flagellum Alginate/Biofilm LPS
Pseudomonas
aeruginosa
Alginate/Biofilm
LPS
Extracellular products
Rhamnolipid
Phospholipase C
Proteases
Siderophores .
Exotoxin A

5. Exotoxin A – The Virulence Factor
Exotoxin A (ETA) is the most potent virulence factor of Pseudomonas aeruginosa
LD50 of 0.2 mg when injected intraperitoneally into a 18-gram mouse
Biological effects
Extensive tissue damage
Promotes bacteria invasion
Interferes with function of the cellular immune system
May lead to systemic disease

6. Exotoxin A – The Enzyme Member of mono-ADP-ribosyl transferase family
Other members include:
Diphtheria toxin, pertussis toxin, cholera toxin, C3 exoenzyme, iota toxin
66 kDa single polypeptide
Three functional domains
Secreted as a proenzyme
Activated within the eukaryotic cell through a proteolytic event
III
Catalytic Ib II
Translocation Ia
Receptor binding
(Wedekind et al., (2001) J. Mol. Biol. 314, 823)

7. Eukaryotic Elongation Factor 2 (eEF2)
Protein substrate for ETA
90 –110 kDa protein
GTPase superfamily
Important factor in the elongation step of protein synthesis
Covalent modification by ETA produces ADP-ribosyl eEF2 (ADPR-eEF2)
Prevents its participation in protein translation
Cell death G′ G II
III V IV
Diphthamide
(Jørgensen et al., (2003) Nat. Struc. Biol. 10, 379)

8. Function of eEF2

9. ADP-Ribosyl Transferase (ADPRT) Reaction

10. Catalytic Domain of ETA (PE24H)
(Li et al., (1996) PNAS 93, 6902)

11. Research Objectives – The Big Picture
General statement
Improve the understanding of the interactions between the catalytic domain of ETA and both its substrates, eEF2 and NAD+
Long term research goals
Understand the detailed reaction mechanism for ETA
Knowledge-based approach to preventing the action of this toxin
Develop new strategies that target ETA to fight Pseudomonas aeruginosa infections

12. Research Objectives – My Specific Projects
Part A: Interactions of the toxin with NAD+
Study of water-soluble inhibitors
Development of a NAD+-glycohydrolase assay
Part B: Toxin-eEF2 interactions
Physiological requirements for binding
Fluorescence-based approach to elucidate sites of contact
Fluorescence resonance energy transfer (FRET) distance study

13. PART A: Interactions of the Toxin with NAD+
Project #1
STUDY OF WATER-SOLUBLE INHIBITORS
Yates, S.P., Taylor, P.L., Jørgensen, R., Ferraris,
D., Zhang, J., Andersen, G.R., and Merrill, A.R.
Biochem. J. (2005) 385:

14. Inhibition of PE24H Previous work from our research group
Characterization of a series of small, non-polar competitive inhibitors
Most potent inhibitor was NAP (1,8-napthalamide)
Model of NAP bound to catalytic domain of ETA
Lack of water-solubility limited the usefulness as potential therapeutic drugs
Armstrong et al., (2002) J. Enzyme Inhib. Med. Chem. 17, 235

15. Aims of Study
Characterize a series of water-soluble compounds for their inhibition against PE24H
Co-crystal structure of the inhibitor PJ34 with PE24H

16. The Inhibitors Mimic nicotinamide
IC50 values ranged from 170 nM to 82.4 mM
GP-D, PJ34, GP-M most potent
Hallmark of a good inhibitor was a planar hetero-ring

17. PJ34 – Further Characterized
Water-soluble phenanthridinone derivative
IC50 = 280 nM
Commercially available
Well-characterized compound
Studied in extensively in several PARP related systems

18. Biochemical Characterization of PJ34
Binding affinity
KD is 820  54 nM
70x tighter binding to PE24H compared to NAD+
Competitive inhibitor
As [PJ34] increases, the KM increases but the Vmax remains unchanged
Ki = 140 nM determined using both Dixon and Lineweaver-Burk methods

19. Crystallization of PE24H-PJ34
Data
2.1 Å resolution
Refinement
R-factor = 21.3 %
Rfree-factor = 23.5 %

20. Hydrophobic Pocket and Active Site
Yates et al., (2005) Biochem. J. 385, 667

21. Interactions in the Active Site
3.1 Å
2.5 Å
2.7 Å
2.5 Å
Yates et al., (2005) Biochem. J. 385, 667

22. Similar Enzymes
Catalytic domain of ETA is functionally and structurally similar to both mono-ADPRTs and PARPs
Diphtheria toxin (DT)
Mono-ADPRT and also catalyzes the ADP-ribosylation of eEF2
PARPs (Poly-(ADP-ribosyl) polymerases)
Catalyzes the covalent attachment of ADP-ribose units to nuclear DNA-binding proteins
Taken from: Putt & Hergenrother (2004) Anal. Biochem.326, 78

23. Comparison to Other Active SitesDT
DT structure:
Bell & Eisenberg, (1996)
Biochemistry 35, 1137
PARP
PARP structure:
Ruf et al., (1998)
Biochemistry 37, 3893

24. Findings for Project #1 Hetero-ring planarity important for inhibition
PJ34 is a competitive inhibitor
First report of a structure of a mono-ADPRT-inhibitor complex
Confirmed the hydrogen bonding of the lactam moiety to Gly-441
Planar compounds sandwich better into the nicotinamide-binding pocket than more flexible compounds
Similarities and differences between bacterial toxins and PARP
Exploit the differences to target one enzyme over the other

25. PART A: Interactions of the Toxin with NAD+
Project #2
DEVELOPMENT OF A
NAD+-GLYCOHYDROLASE ASSAY
Yates, S.P., and Merrill, A.R. Anal. Biochem. (2005) in press.

26. NAD+-Glycohydrolase Activity2 C
CH2 N P
NH2 - +
STEP 1
nicotinamide
STEP 2
ADP-ribose
NAD+
oxacarbenium ion

27. F-NAD+ Initial inhibitor study showed that IC50 value is 82.4  7.4 M
Binding affinity to toxin similar to NAD+
NAD+  KD = 53  2 M
F-NAD+  KD = 33  1 M

28. Aims of Study
Is F-NAD+ a competing substrate or a competitive inhibitor?
Is the C-N bond broken?
Develop an HPLC-based NAD+-glycohydrolase assay
Why?
Fluorometric assay uses e-NAD+
Contains a etheno bridge which gives rise to its fluorescence
F-NAD+ lacks this structural feature

29. Reaction and Sample Preparation
Samples (25 mL) taken
at t = 0 to 4 hrs
Reaction Setup
Toxin + NAD+ (250 mL)
Sampling
Inhibit Reaction
To HPLC
PE24H bound to resin
Load to Spin Column
Toxin Removed
Add 75 mL
Mobile Phase
(with internal standard)
Chelating Sepharose
Spin Column
Flow-Through
ready for HPLC –
contains no protein

30. HPLC Instrumentation Setup
Inject sample via sample loop
150 mm
Precolumn
C18 column – reverse phase
4.6 mm
Detector at 259 nm
Mobile phase: 20 mM NaHPO4, pH 5.5: acetonitrile (100:5 v/v %)

31. HPLC and Analysis – Rate Determination+
Chromatogram
Nicotinamide Standard Curve
Time Course Plot
Rate = 55  3 mM nicotinamide produced per hour

32. Rate of Hydrolysis of F-NAD+
Visual inspection of chromatograms shows the peak area for ADPR increasing
Mathematically deconvoluted ADPR peak from NAD+ or F-NAD+ peak
Hydrolysis of F-NAD+ is 0.2% rate of NAD+

33. Findings for Project #2 HPLC-based NAD+-glycohydrolase assay developed
Addition of spin column step allows quick removal of protein
F-NAD+ binds to the enzyme but not readily hydrolyzed
What does fluorine substitution at 2'-OH position do?
Disrupts hydrogen bond between Glu-553 and 2'-OH position
This hydrogen bond important for bond breakage
Cause nicotinamide leaving group to depart slower
Fluorine substituent may destabilize cationic intermediate

34. PART B: Toxin-eEF2 Interactions
Project #3
PHYSIOLOGICAL REQUIREMENTS FOR BINDING
Loop
Yates, S.P., and Merrill, A.R. J. Biol. Chem. (2001) 276:
pH and Guanyl nucleotide
Armstrong, S., Yates, S.P., and Merrill, A.R. J. Biol. Chem. (2002) 277:
ADPR-eEF2
Jørgensen, R., Yates, S.P., Teal, D.J., Nilsson, J., Prentice, G.A., Merrill, A.R., and Andersen, G.R.
J. Biol. Chem. (2004) 279:

35. Aims of Study
Investigate the conditions required for toxin-eEF2 interaction
Effect of pH
Effect of bound guanyl nucleotides on eEF2
Effect of ADP-ribosylation of eEF2
Functional role of a surface-exposed loop near the active site

36. FRET-based eEF2 Binding Assay
Fluorescence Resonance Energy Transfer (FRET)
Transfer of excitation energy from a donor fluorophore to a an acceptor fluorophore through non-radiative dipole-dipole interactions
Criteria
Donor and acceptor in close proximity
Acceptor absorption overlaps with fluorescence emission of donor
Dipole-dipole interactions are parallel
Donor fluorophore
PE24H labelled with IAEDANS (PE24H-AEDANS)
Acceptor fluorophore
eEF2 labelled with fluorescein (eEF2-AF)

37. Effect of pH on eEF2 Binding to Toxin
Optimum eEF2 binding at pH 7.8
Two distinct pKa values
Acidic pKa = 6.3
His residue
Alkaline pKa = 9.3
Tyr residue
pH profiles for eEF2 binding and catalysis very similar
eEF2 binding may be responsible for pH dependence observed in catalysis

38. Effect of Guanyl Nucleotides
eEF2 is a member of the GTPase superfamily
Does the toxin require a specific eEF2 conformation for binding?
eEF2 with non-hydrolyzable GTP/GDP analogues bound
Toxin does not prefer a specific state of eEF2 for either binding or catalytic function

39. Interaction of ADPR-eEF2 with Toxin
ADPR-eEF2 maintained the ability to bind toxin
Active site of toxin can accommodate the bulky ADP-ribose group
Structures of both eEF2 and ADPR-eEF2 recently solved
No major conformational changes induced after ADP-ribosylation

40. Characterization of a Loop in ETA
History of Loop C
Residues
Functional removal
Decreases activity significantly (1.8 x 10+4-fold)
Retains ability to bind NAD+ near wild-type levels
Alanine-scanning mutagenesis
Some mutant proteins exhibited reduced activity
KD and KM for NAD+ similar to wild-type
What is the role of this Loop?
Catalytic or eEF2 substrate binding?
Loop C
(Li et al., (1996) PNAS 93, 6902)

41. Determination of KM and KD for eEF2
Alanine-scanning mutants
KM for eEF2 unaffected
Enzyme rate (kcat) is affected
pG-Loop C mutant protein
Each residue within Loop C replaced with glycine
Functional removal of loop
Retained ability to associate with eEF2 at normal levels
Loop is a catalytic element
May modulate the transferase activity of the toxin

42. Findings for Project #3 Toxin-eEF2 association is pH-dependent
Correlates to that observed for catalytic function
GTP or GDP bound to eEF2 did not affect it as a protein substrate
Structurally the diphthamide and guanyl nucleotide binding site are quite distant
No direct coupling of sites
Toxin maintains the ability to associate with eEF2 after its ADP-ribosylation
Loop C is important for catalysis
May stabilize the transition state structure during the catalytic reaction

43. PART B: Toxin-eEF2 Interactions
Project #5
FLUORESCENCE-BASED APPROACH TO
ELUCIDATE SITES OF CONTACT
Yates, S.P., and Merrill, A.R. Biochem. J. (2004) 379:

44. Aim of Study Identify contact sites between eEF2 and PE24H
This protein-protein interaction is poorly characterized
Two extreme models are possible
Minimal Contact Model  Maximum Contact Model
PE24H
eEF2
eEF2
PE24H

45. Experimental Approach
Single cysteine residues introduced into PE24H at 21 defined surface sites and labelled with the fluorophore, IAEDANS O
IAEDANS
NHCH CH NH 2 2 C CH I 2 .. HS CH
PROTEIN 2 SO 3 H O
NHCH CH NH C CH S CH
PROTEIN 2 2 2 2
Protein adduct
(Li et al., (1996) PNAS 93, 6902) + HI SO H 3

46. Experimental Approach
Fluorescence studies performed in the presence and absence of eEF2
Fluorescence wavelength emission maxima (lem,max)
Fluorescence lifetime
Acrylamide quenching

47. Fluorescence lem,max and Lifetime

48. Acrylamide Quenching
Measure the ability of acrylamide to quench the fluorescence of IAEDANS probe attached to PE24H
Acrylamide is a water-soluble, non-ionic quencher
The more accessible the probe is to acrylamide, the more quenching is observed
Determine the bimolecular quenching constant (kq) in the presence and absence of eEF2 using the Stern-Volmer equation
kq is the rate of collisions with the quencher that result in deactivation of excited state of the fluorophore
F0/F
[Q] 1

49. Acrylamide Quenching
50%

50. Crude Model of PE24H-eEF2 Complex
Potential eEF2 contact sites on PE24H
Minimal contact between proteins
Diphthamide residue on eEF2 positioned near scissile glycosidic bond of NAD+ in active site 1 2 3 4 5 6 7 8 9
PE24H
Domain IV
of eEF2
diphthamide
507
554
519
459
449
442
486
410
408
(Li et al., (1996) PNAS 93, 6902;
Jørgensen et al., (2003) Nat. Struc. Biol. 10,379)

51. Findings for Project #4
Fluorescence lem,max and lifetime suggested minimal contact
No large changes observed after eEF2 complexation
Probes near active site or catalytic loop showed greatest change in acrylamide quenching after eEF2 binding
Other locations showed smaller changes in kq
A crude toxin-eEF2 model was proposed
Contact between PE24H and eEF2 is minimal

52. PART B: Toxin-eEF2 Interactions
Project #5
FRET DISTANCE STUDY

53. Aim of Study Better define the proposed minimal contact model
Measure the distances between selected residues in PE24H to eEF2 using FRET
Design and create recombinant mutant proteins of eEF2 to serve as the acceptor fluorophore reference

54. Mutant eEF2 Proteins – Selection
Introduce a cysteine into domain IV at a defined location to conjugate the fluorescein probe
Thr-574 and Thr-812 chosen sites to mutate
Non-conserved residues
Surface exposed side chains
Estimated that these residues will be an ideal distance to PE24H
Thr-812
Thr-574
Diphthamide
(Jørgensen et al., (2003) Nat. Struc. Biol. 10,379)

55. Mutant eEF2 Proteins - Creation
Site-directed mutagenesis to create desired mutation
Introduce plasmid into Saccharomyces cerevisiae
Select for strain expressing the recombinant mutant eEF2
His-tag purification
T812C-yeEF2H protein is unstable
T574C-yeEF2H purifies at levels similar to wild-type

56. FRET Approach PE24H-AEDANS (donor) eEF2-AF (acceptor)
Calculate distance between donor and acceptor using a series of equations J

57. FRET between Toxin and T574C-eEF2

58. Anisotropy
Measures local rotational motion of the IAEDANS probe on PE24H before and after eEF2 complexation
Do any of the probes have significantly restricted mobility after eEF2 binds?
Can we assume that k2 is two-thirds?
N577C-AEDANS
After eEF2 associates this probe displays significantly hindered mobility

59. Development of FRET Distance Model
Important to remember
The apparent distances have % uncertainty
Length of linker for probes contributes to distance
Efficiency depends on the orientation of the probes
Position fluorescein probe on eEF2 in three-dimensional space to best satisfy calculated distances
T574C-AF
Cys-AEDANS
Cys-AF
585
525
592
577
486
490
507
410
(Li et al., (1996) PNAS 93, 6902)

60. Comparison to X-ray Structure of Complex
Does this FRET model agree with the recently solved toxin-eEF2 structure?
(Jørgensen et al., manuscript in preparation)

61. Effect of -TAD (An NAD+-Analogue)
585
577
525
592
486
490
507
410
(Li et al., (1996) PNAS 93, 6902)

62. Findings for Project #5
FRET-based model and X-ray structure agree within the error of the technique
N577C-AEDANS is the exception
Anisotropy values suggests probe restriction
Distances shorten between the toxin and eEF2 when -TAD is bound in the complex

63. Earlier Crude Model vs. X-ray Structure
eEF2
PE24H
(from Project #4)
(Jørgensen et al., manuscript in preparation)
PE24H

64. Final Thoughts
Improved understanding of structural features important for inhibition
Hetero-ring planarity
X-ray structure of inhibitor with toxin
Able to distinguish a substrate from an inhibitor
HPLC-based NAD+-glycohydrolase assay allows direct observation of products
Toxin highly adaptable
Ability to bind eEF2 and its many forms (GTP/GDP, ADPR)
pH dependence for catalysis now assigned to eEF2 binding
Minimal contact model best describes toxin-eEF2 interactions
FRET distance model correlates with X-ray structure

65. Acknowledgements Supervisor Merrill Research Group
Dr. Rod Merrill Trish Taylor, Gerry Prentice,
Abdi Musse
Univ. of Aarhus, Denmark Guilford Pharmaceuticals
Dr. Gregers R. Andersen Dr. Jie Zhang
René Jørgensen Dr. Dana Ferraris
Advisory Committee Examination Committee
Dr. Joe Lam Dr. Dan Thomas
Dr. Bob Keates Dr. Joe Lam
Dr. John Honek, Univ. of Waterloo Dr. Michael Palmer, Univ. of Waterloo
Dr. Jean Gariépy, Univ. of Toronto
Univ. of California, San Francisco University of Guelph
Dr. Norman Oppenheimer Dr. Adrian Schwan
Financial Support Family and Friends
Canadian Cystic Fibrosis Foundation Parents
PhD CCFF Studentship Matthew Davidson
Canadian Institutes of Health Research

66. My PhD Journey!
COMPS!