Transcript seminar_examination.p - University of Guelph
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
Outline
Background
Research Objectives
Inhibition of the catalytic domain of exotoxin A Interactions between the toxin and its protein substrate
Final Thoughts
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
Virulence Factors
Flagellum
Pseudomonas aeruginosa
Alginate/Biofilm Extracellular products Rhamnolipid Phospholipase C Proteases .
Siderophores Exotoxin A Pilus LPS
Exotoxin A – The Virulence Factor
Exotoxin A (ETA) is the most potent virulence factor of
Pseudomonas aeruginosa
LD 50 of 0.2 mouse m g when injected intraperitoneally into a 18-gram Biological effects Extensive tissue damage Promotes bacteria invasion Interferes with function of the cellular immune system May lead to systemic disease
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
Ib III Catalytic II Translocation Ia Receptor binding
(Wedekind et al., (2001) J. Mol. Biol. 314, 823)
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
II Diphthamide G′ III IV G V
(J ørgensen et al., (2003) Nat. Struc. Biol. 10, 379)
Function of eEF2
ADP-Ribosyl Transferase (ADPRT) Reaction
H 2 N
NAD +
N N N N O H 2 C
A-phosphate
O P O O O P O O O -
N-phosphate
CH 2 O + N O NH 2
A-ribose
HO OH O HO H OH
N-ribose
N H 2 N N
A-ribose
HO N
STEP 1
NH 2 N
nicotinamide A-phosphate
N O H 2 C O O P O O O O P O
N-phosphate
CH 2 O + H H 2 N OH N HO OH
oxacarbenium ion STEP 2 -H +
H O N
N-3
N H C CH 2 H 2 C
diphthamide residue of eEF2
NH CH 2 + N(CH 3 ) 3 CH O H 2 N N N
A-ribose
HO N O H 2 C O P O O O P O O O CH 2 O H H NH C CH 2 O OH HO
N-ribose ADP-ribosyl - eEF2
N OH H 2 C CH 2 N + N(CH 3 ) 3 CH O H 2 N
Catalytic Domain of ETA (PE24H)
-TAD Tyr-470 Tyr-481 His-440 Glu-553
(Li et al., (1996) PNAS
93,
6902)
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
Research Objectives – My Specific Projects
Part A : Interactions of the toxin with NAD + 1.
2.
Study of water-soluble inhibitors Development of a NAD + -glycohydrolase assay Part B : Toxin-eEF2 interactions 3.
4.
5.
Physiological requirements for binding Fluorescence-based approach to elucidate sites of contact Fluorescence resonance energy transfer (FRET) distance study
PART A: Interactions of the Toxin with NAD
+ Project #1 STUDY OF WATER-SOLUBLE INHIBITORS
Yates, S.P., Taylor, P.L., J ørgensen, R., Ferraris, D., Zhang, J., Andersen, G.R., and Merrill, A.R. Biochem. J. (2005) 385:667-675.
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
Aims of Study
Characterize a series of water-soluble compounds for their inhibition against PE24H Co-crystal structure of the inhibitor PJ34 with PE24H
The Inhibitors
Mimic nicotinamide IC 50 values ranged from 170 nM to 82.4 m M GP-D, PJ34, GP-M most potent Hallmark of a good inhibitor was a planar hetero-ring O NH NH H 3 C O N CH 3
PJ34
NH O NH N F N
Tricyclic Lactams – [6,6,6]-Ring System
O O O NH NH NH O N N N N N O N
GP-L
N CH 3
GP-G
H 3 C N
GP-N Tricyclic Lactams – [5,6,7]-Ring System
O O NH NH CH 3 N N N N N CH 3 CH 3 O
GP-D Bicyclic Lactam
O NH + NH 3 Cl -
5-AIQ
H 3 C N CH 3
GP-F Tetracyclic Lactam
O NH O
GP-P
SO 3 H O NH N H N O N
GP-M
O NH N N N
GP-H GP-I NAD + Analogue
O NH 2 N + O NH 2 F N N O O P O OH O O P O O O N N OH OH
2’-F-ribo-NAD + (F-NAD + )
N
PJ34 – Further Characterized
Water-soluble phenanthridinone derivative IC 50 = 280 nM Commercially available Well-characterized compound Studied in extensively in several PARP related systems O NH NH H 3 C N CH 3 O
Biochemical Characterization of PJ34
Binding affinity K D is 820 54 nM 70x tighter binding to PE24H compared to NAD + Competitive inhibitor As [PJ34] increases, the K M increases but the V max remains unchanged K i = 140 nM determined using both Dixon and Lineweaver-Burk methods 1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 1000 2000 3000
[PJ34], nM
4000 5000 0.4
0.3
0.2
0.1
0.0
-0.1
1.0
0.9
0.8
0.7
0.6
0.5
-200 50 m M -NAD + 100 m M -NAD + 200 m M -NAD + 300 m M -NAD + 500 m M -NAD + 0 200 400 600
[PJ34], nM
800 1000 1200
Crystallization of PE24H-PJ34
Data
2.1 Å resolution
Refinement
R-factor = 21.3 % R free -factor = 23.5 %
Hydrophobic Pocket and Active Site
Yates et al., (2005) Biochem. J. 385 , 667
Interactions in the Active Site
3.1 Å 2.7 Å 2.5 Å 2.5 Å
Yates et al., (2005) Biochem. J. 385 , 667
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
Comparison to Other Active Sites
DT
DT structure: Bell & Eisenberg, (1996) Biochemistry 35 , 1137
PARP
PARP structure: Ruf et al., (1998) Biochemistry 37 , 3893
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
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.
NAD
+
-Glycohydrolase Activity
H 2 N N N H 2 N N N HO N N O H 2 C O P O O O P O O O CH 2 O + N OH
STEP 1
HO
NAD +
O NH 2 N
nicotinamide
H OH N N O H 2 C O O P O O P O O O CH 2 O + H O NH 2 O H + H + HO OH HO OH
oxacarbenium ion
H 2 N
STEP 2 - H +
N N N HO N O H 2 C O O P O O P O O O CH 2 O OH HO H OH OH
ADP-ribose
F-NAD
+ Initial inhibitor study showed that IC 50 value is 82.4 7.4 m M Binding affinity to toxin similar to NAD + NAD + F-NAD + K D K D = 53 = 33 2 m M 1 m M O N + O O O P O OH O O P O O F O NH 2 N N OH NH N OH 2 N 1.0
0.8
0.6
0.4
0.2
0.0
0 F-NAD + NAD + 200 400 600 [NAD + or F-NAD + ], m M 800 1000
A.
NAD +
H 2 N N N N N O H 2 C
-phosphate
O P O O O O P O O
-phosphate
CH 2 O + N
A-ribose
HO OH HO H OH
N-ribose
O H 2 N N
STEP 1
NH 2 N
nicotinamide
N
-phosphate
O O N A.
H 2 N N O H 2 C P O O O P O O
-phosphate
CH 2 O + H N O OH + H +
NAD +
N N N O H 2 C
-phosphate
O P O O O O P O O
-phosphate
CH 2 O + N NH 2 HO
A-ribose
OH H 2 N HO OH
oxacarbenium ion STEP 2 - H +
H 2 O
A-ribose
HO OH O HO H OH
N-ribose
N
-phosphate
OH + H + N NH 2 H 2 N
STEP 1
N N
nicotinamide
-phosphate
N O O N N H 2 C O P O O P O O
-phosphate
CH 2 O + O
Aims of Study
HO OH HO OH H N
A-ribose
HO N O H 2 C OH O P O O O O P O O CH 2
-phosphate
O H HO
N-ribose
OH OH
ADP-ribose
A-ribose
Is F-NAD +
oxacarbenium ion
a competing substrate or a competitive inhibitor?
N H 2 N Is the C-N bond broken?
N
A-ribose
Why?
HO N N H 2 C
-phosphate
OH
STEP 2
O O O P
-phosphate
F-NAD + O O P O -
- H +
O CH 2 HO O H OH OH B.
Develop an HPLC-based NAD + -NAD + H 2 N N lacks this structural feature N
A-ribose
HO N N O H 2 C OH
-phosphate
-glycohydrolase assay O P O O O P O O
-phosphate
CH 2 HO O F + N H
N-ribose F-NAD + 2'-F-N-ribose
Contains a etheno bridge which gives rise to its fluorescence
ADP-ribose
O NH 2 O NH 2 B.
H 2 N N N
A-ribose
HO N N O H 2 C
-phosphate
O P O O O O P O O
-phosphate
CH 2 O OH + N H HO F
2'-F-N-ribose F-NAD +
O C.
NH 2 NH N N
-A-ribose
HO + N N O H 2 C
-phosphate
O O O P O O P O O
-phosphate
CH 2 O OH + N HO
N-ribose
H OH
-NAD +
O NH 2 C.
NH N N
-A-ribose
HO + N N O H 2 C
-phosphate
O O O P O O P O O
-phosphate
CH 2 O OH + N HO
N-ribose
H OH
-NAD +
O NH 2
Reaction and Sample Preparation
Samples (25
m
L) taken at t = 0 to 4 hrs Sampling Reaction Setup Toxin + NAD + (250
m
L) Load to Spin Column Add 75
m
L Mobile Phase (with internal standard) Toxin Removed Chelating Sepharose Spin Column PE24H bound to resin Flow-Through ready for HPLC – contains no protein
HPLC Instrumentation Setup
Inject sample via sample loop Precolumn 150 mm C18 column – reverse phase 4.6 mm Detector at 259 nm
Mobile phase: 20 mM NaHPO 4 , pH 5.5: acetonitrile (100:5 v/v %)
HPLC and Analysis – Rate Determination
0.10
0.8
NAD +
0.08
0.6
0.06
0.04
0.02
0.00
ADPR + PABA nicotinamide
0.4
0.2
0.0
0 100 200 300 pmoles of nicotinamide 400 500 0 2 4 6 Retention Time (minutes) 8
Nicotinamide Standard Curve Chromatogram
300 250 200 150 100 50 0 0 1 2 Time (hours) 3 4
Time Course Plot Rate = 55
3
m
M nicotinamide produced per hour
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 + 0.2% rate of NAD + is 0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.06
0.05
0 0.04
ADPR F-NAD + PABA
2 4 6 Retention Time (minutes)
F-NAD + nicotinamide
0 hours 8 48 hours 0.03
0.02
0.01
0.00
ADPR PABA nicotinamide
0 2 4 6 Retention Time (minutes) 8
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
PART B: Toxin-eEF2 Interactions
Project #3 PHYSIOLOGICAL REQUIREMENTS FOR BINDING
Loop Yates, S.P., and Merrill, A.R. J. Biol. Chem. (2001) 276:35029-35036.
pH and Guanyl nucleotide Armstrong, S., Yates, S.P., and Merrill, A.R. J. Biol. Chem. (2002) 277:46669-46675.
ADPR-eEF2 J ørgensen, R., Yates, S.P., Teal, D.J., Nilsson, J., Prentice, G.A., Merrill, A.R., and Andersen, G.R. J. Biol. Chem. (2004) 279:45919-45925.
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
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 Fluorescence Acceptor Absorption
Wavelength (
)
Donor fluorophore PE24H labelled with IAEDANS ( PE24H-AEDANS ) Acceptor fluorophore eEF2 labelled with fluorescein ( eEF2-AF )
Effect of pH on eEF2 Binding to Toxin
Optimum eEF2 binding at pH 7.8
Two distinct pK
a
values Acidic pK
a
= 6.3
His residue Alkaline pK
a
= 9.3
Tyr residue pH profiles for eEF2 binding and catalysis very similar eEF2 binding may be responsible for pH dependence observed in catalysis
5.0
4.0
3.0
2.0
1.0
0.0
9.0
8.0
7.0
6.0
4 6 8 pH 10 12
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
1.0
0.8
0.6
0.4
eEF2 substrate Native-absence of bound nucletides GDP -S bound GTP -S bound Relative ADPRT 100 9 a 102 4 92 10
0.2
0.0
0
eEF2-AF GTP -S-eEF2-AF GDP -S-eEF2-AF
1000 2000 3000 4000 [eEF2-AF], nM 5000 6000
Toxin does not prefer a specific state of eEF2 for either binding or catalytic function
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
1.0
0.8
0.6
0.4
0.2
0.0
0 1000 2000
eEF2-AF ADPR-eEF2-AF
3000 [eEF2-AF] or [ADPR-eEF2-AF], nM 4000
Characterization of a Loop in ETA
History of Loop C Residues 483-490 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 K D and K M wild-type for NAD + similar to What is the role of this Loop?
Catalytic or eEF2 substrate binding?
Loop C
-TAD Tyr-481 His-440 Tyr-470 Glu-553
(Li et al., (1996) PNAS
93,
6902)
Determination of K
M
and K
D
for eEF2
Wild-type Q483A D484A Q485A D488A Relative
k cat
1.00 0.05 0.11 0.01 0.07 0.003 0.69 0.01 0.17 0.01 Relative
K
M(eEF2) 1.00 0.12 1.02 0.09 2.19 0.21 1.01 0.05 2.06 0.13 Relative specificity Alanine-scanning mutants 1.00 0.07 0.11 0.68 K 0.03 0.002 0.02 0.08 0.001 M for eEF2 unaffected cat ) is affected
1.0
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
0.8
0.6
0.4
0.2
wild-type PE24H pG-Loop-C PE24H
0.0
0 1000 2000 3000 [eEF2-AF], (nM)
Loop is a catalytic element May modulate the transferase activity of the toxin
4000 5000
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
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) 379:563-572.
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 PE24H eEF2
Experimental Approach
Single cysteine residues introduced into PE24H at 21 defined surface sites and labelled with the fluorophore, IAEDANS
O NHCH 2 CH 2 NH C CH 2 I IAEDANS Ala-519 Gly-525 Gln-603 Ser-507 Gly-549 Ser-515 Ala-476 Glu-486 Arg-490 Thr-442 Thr-554 Ser-459
-TAD Thr-564 Gln-592 Ser-449 Ser-585 Asn-577 Ser-410 Ser-408 Gln-415 Gln-428
(Li et al., (1996) PNAS
93,
6902)
SO 3 H ..
HS CH 2 PROTEIN O SO 3 H NHCH 2 CH 2 NH C CH 2 S CH 2 PROTEIN Protein adduct
+ HI
Experimental Approach
Fluorescence studies performed in the presence and absence of eEF2 Fluorescence wavelength emission maxima ( em,max ) Fluorescence lifetime Acrylamide quenching
Fluorescence
em,max
and Lifetime
Protein adduct 1 S408C 2 3 4 5 6 7 8 S410C Q415C Q428C T442C S449C S459C A476C 9 E486C 10 R490C 11 S507C 12 S515C 13 A519C 14 G525C 15 G549C 16 T554C 17 T564C 18 N577C 19 S585C 20 Q592C 21 Q603C 0 (ns) – eEF2 13.8 0.4 14.4 0.1 14.3 0.3 15.9 0.5 16.9 0.5 15.2 0.1 14.4 0.5 15.9 0.2 14.9 0.1 12.0 0.5 13.2 0.2 16.0 0.2 15.7 0.1 13.4 0.1 15.5 0.4 15.3 0.1 16.7 0.3 12.7 0.1 14.0 0.2 15.2 0.4 14.2 0.1 + eEF2 13.6 0.5 14.5 0.2 13.9 0.3 14.7 0.1 16.8 0.4 15.7 0.9 13.9 0.5 15.6 0.8 14.6 0.6 11.7 0.3 13.7 0.4 15.4 0.3 16.9 0.1 13.4 0.3 15.8 0.2 15.9 0.8 16.5 0.2 12.4 0.4 14.2 0.6 14.9 0.6 14.0 0.7 em,max (nm) – eEF2 481 479 478 478 471 473 479 478 478 483 482 473 479 481 480 478 474 482 481 478 481 + eEF2 481 479 479 479 472 477 480 478 479 483 481 476 478 481 479 477 475 482 481 479 482
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 (k
q
) in the presence and absence of eEF2 using the Stern-Volmer equation k
q
is the rate of collisions with the quencher that result in deactivation of excited state of the fluorophore F 0 /F 1
F
0
F
K SV
[
acrylamide
] + 1
K SV
[Q] 0
k q
Acrylamide Quenching
Protein adduct 1 2 3 4 5 6 7 8 S408C S410C Q415C Q428C T442C S449C S459C A476C 9 E486C 10 R490C 11 S507C 12 S515C 13 A519C 14 G525C 15 G549C 16 T554C 17 T564C 18 N577C 19 S585C 20 Q592C 21 Q603C
k q
(x 10 9 M -1 s -1 ) – eEF2 1.03 0.02 1.29 0.01 1.28 0.04 0.86 0.03 0.56 0.02 0.60 0.02 0.94 0.03 0.89 0.03 1.29 0.04 1.27 0.04 0.80 0.02 0.55 0.03 1.23 0.04 0.96 0.01 0.65 0.03 0.88 0.04 0.50 0.02 1.10 0.04 0.90 0.02 1.42 0.02 0.68 0.02 + eEF2 0.37 0.01 0.36 0.01 0.73 0.01 0.54 0.01 0.20 0.01 0.27 0.01 0.42 0.02 0.58 0.01 0.48 0.02 0.70 0.02 0.33 0.01 0.41 0.01 0.61 0.02 0.75 0.02 0.35 0.01 0.16 0.01 0.27 0.01 0.74 0.01 0.59 0.01 0.79 0.01 0.39 0.01
6.0
5.5
5.0
2.5
2.0
1.5
1.0
4.5
4.0
3.5
3.0
*
Residue Number 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
554 2 6 442
(Li et al., (1996) PNAS
93,
6902; J ørgensen et al., (2003) Nat. Struc. Biol. 10, 379)
Findings for Project #4
Fluorescence em,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 k
q
A crude toxin-eEF2 model was proposed Contact between PE24H and eEF2 is minimal
PART B: Toxin-eEF2 Interactions
Project #5 FRET DISTANCE STUDY
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
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
(J ørgensen et al., (2003) Nat. Struc. Biol. 10, 379)
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
FRET Approach
PE24H-AEDANS (donor) eEF2-AF (acceptor) Calculate distance between donor and acceptor using a series of equations
E
1
F DA F D R
0 9 .
8 10 3
J
2
Q D n
4 1 / 6 Å
R
R
0
E
1 1 1 / 6 Donor Fluorescence Acceptor Absorption
J
Wavelength (
)
FRET between Toxin and T574C-eEF2
PE24H AEDANS Adducts S410C E486C R490C S507C G525C N577C S585C Q592C Quantum Yield 0.41 0.43 0.31 0.33 0.33 0.33 0.40 0.38 Overlap Integral,
J
(x 10 -13 cm 3 M -1 ) 1.72372 1.72956 1.80256 1.78248 1.75769 1.75784 1.83424 1.82695 Förster Distance
R
0 (Å) 47.0 47.4 45.2 45.7 45.6 45.6 47.3 47.0 Efficiency (%) 40.9 32.0 24.3 30.3 28.3 22.9 27.3 49.3 Estimated Distance
R
(Å) 49.9 53.8 54.7 52.5 53.3 55.8 55.7 47.2
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 2 is two-thirds?
PE24H-AEDANS Adducts S410C E486C R490C S507C G525C N577C S585C Q592C (–) yeEF2 0.086 0.001 0.106 0.002 0.066 0.003 0.075 0.003 0.079 0.002 0.074 0.002 0.078 0.004 0.087 0.002 (+) yeEF2 0.093 0.001 0.113 0.001 0.080 0.003 0.110 0.001 0.097 0.001 0.135 0.001 0.106 0.001 0.129 0.001 % increase 7.0 6.0 17.1 32.3 18.4 45.0 25.9 32.5 N577C-AEDANS After eEF2 associates this probe displays significantly hindered mobility
Development of FRET Distance Model
Important to remember The apparent distances have 10-20% uncertainty Length of linker for probes contributes to distance
Cys-AEDANS Cys-AF T574C-AF 585 525 592
Efficiency depends on the orientation of the probes Position fluorescein probe on eEF2 in three-dimensional space to best satisfy calculated distances
507 410 577 486 490
(Li et al., (1996) PNAS
93,
6902)
Comparison to X-ray Structure of Complex
Does this FRET model agree with the recently solved toxin eEF2 structure?
(J ørgensen et al., manuscript in preparation)
Effect of
-TAD (An NAD
+
-Analogue)
525 507 592 585 410 577 486 490
(Li et al., (1996) PNAS
93,
6902) PE24H-AEDANS Adducts G525C - -TAD G525C + -TAD N577C - N577C + -TAD -TAD Efficiency (%) 28.3 41.6 22.9 31.1 Estimated Distance
R
(Å) 53.3 48.5 55.8 52.3
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
Earlier Crude Model vs. X-ray Structure
eEF2 (from Project #4) PE24H
PE24H
(J ørgensen et al., manuscript in preparation)
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
Acknowledgements
Supervisor
Dr. Rod Merrill
Univ. of Aarhus, Denmark
Dr. Gregers R. Andersen René Jørgensen
Advisory Committee
Dr. Joe Lam Dr. Bob Keates Dr. John Honek, Univ. of Waterloo
Univ. of California, San Francisco
Dr. Norman Oppenheimer
Financial Support
Canadian Cystic Fibrosis Foundation PhD CCFF Studentship Canadian Institutes of Health Research
Merrill Research Group
Trish Taylor, Gerry Prentice, Abdi Musse
Guilford Pharmaceuticals
Dr. Jie Zhang Dr. Dana Ferraris
Examination Committee
Dr. Dan Thomas Dr. Joe Lam Dr. Michael Palmer, Univ. of Waterloo Dr. Jean Gari épy, Univ. of Toronto
University of Guelph
Dr. Adrian Schwan
Family and Friends
Parents Matthew Davidson