Transcript The Application of Spectrally-Enhanced Proteins to the

Shining Light on the ADPRibosylation Mechanism
of Pseudomonas Toxin
A.R. Merrill
Dept. of Chem. & Biochem.
Univ. of Guelph
Guelph, Ontario, Canada
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P. aeruginosa

(Dennis Kunkel; Microscopy, 2001)
ubiquitous, Gram-neg
bacterium--Pathogenic
 cystic fibrosis, cancer,
burn, AIDS, and postoperative patients
 infections: acute localized
to systemic
 leukopenia, circulatory
collapse, liver, kidney, and
skin necrosis,
hemorrhaging, corneal
destruction, and
pneumonia
 most virulent factor-Exotoxin A
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Exotoxin A
 66
kDa protein secreted by P. aeruginosa
 LD50 = 0.2 mg/kg (mice)
 mono-ADPRT enzyme
• related to diphtheria, cholera, tetanus,
pertussis toxins, PARPs
 cellular
effect: inhibition of protein
synthesis by alteration of elongation
factor 2 (eEF2)
4
Intoxication Mechanism
H+
Intact ETA
28 kDa fragment
37 kDa fragment
ETA Receptor
Furin-like enzyme
Disulfide bond
5
Crystal Structure of ETA
Domain Ib
Domain II
Domain Ia
Domain III
(Wendekind et al., JMB 2001)
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ADPRT Reaction
NAD+
eEF2 diphthamide residue
+ H+
nicotinamide
ADP-ribosylated eEF2 diphthamide residue
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Catalytic Domain of ETA (PE24)
-TAD
His440
Tyr470
Tyr481
Glu553
(Li et al (1996) PNAS 93:6902)
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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 peptidyltRNA on the ribosome in
protein elongation
17.5Å EM Structure
tRNA
Gomez-Lorenzo et al., 2000
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Structure of Yeast eEF2
Diphthamide
IV
(Jorgensen et al., Nat. Struct.
Biol. 10, 387-385, 2003)
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Regulation of eEF2

A diphthamide residue is the
site of regulatory modification
on eEF2
O
NH
N
+N(CH )
3 3


eEF2  a substrate for cellular
ADPRTs which function to
regulate protein synthesis as
part of normal metabolism
NH
O
H2N
diphthamide
Bacterial toxins exploit the existing system
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ADPRT Reaction
NAD+
E
eEF2
E-NAD+
+
E + NAD
eEF2~ADPR
E-NAD+ ~eEF2
k1
k -1
+ k2
E-NAD
k -2

E-Nic~eEF2~ADPR
+
E-NAD ~eEF2
k3
k -3
Nic + H+
E-Nic
E
E-Nic~eEF2~ADPR
k4
k -4
E-Nic
k5
k -5
eEF 2~ADPR
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Principles of Fluorescence
S2
S1
Fluorescence
Absorption
S0
EXCITATION
EMISSION
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Protein Fluorescence
Tyr
Phe
Trp
(Lakowicz, 1983)
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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 p electrons of indole ring
• 1La and 1Lb transitions
• dipole
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ETA Kinetic Parameters
Substrate
Parameter
NAD+
eEF2
KM (mM)
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.
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Steady State Fluorescence

Change in fluorescence intensity as a function of
substrate/ligand concentration
1.0 -
Fi/ Fmax
Fi
[ligand]
[ligand]
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NAD Binding
1.2
1.0
0.6
(F/Fmax)
Fractional Saturation
0.8
Kd=50 mM
0.4
0.2
A.
0.0
0
200
400
600
800
1000
2.25
2.20
Fluorescence (volts)
-1
kobs (s )
600
500
2.15
2.10
2.05
2.00
1.95
1.90
1.85
0.005
400
Armstrong & Merrill,
Biochemistry, in press
0.010
0.015
0.020
Time (seconds)
300
B.
200
0
10
20
30
40
50
60
70
80
90
+
[NAD ] mM
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Stopped Flow Fluorescence
 Kinetic
data fit to exponentials:
• Single exponential
F  F0  F1 (1  e  kt )
• Multiple exponential
F  F0  F1 (1  e  k1t )  F2 (1  e  k 2 t )  ...
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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 (mM-1s-1)
4.7  0.4
320  39
82 ± 9
koff (s-1)
194 ± 15
131 ± 22
51 ± 6
koff/kon (mM)
41 ± 3
0.41 ± 0.10
0.62 ± 0.07
Kd (mM)
45 ± 5
0.71 ± 0.21 0.054 ± 0.006
1.2 ± 0.1
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Ala Scan of Loop C (483-490)
Ala
Arg
1.0
0.8
Relative k cat
Glu
0.6
*
Pro
0.4
*
Gln
0.2
Yates & Merrill (2001)
*
* Asp
*
JBC 276,35029
Asp
Gln
0.0
483
484
485
486
487
488
489
490
Residue Number
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eEF2 Docking Site
Loop C
PE24
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Preparation of AEDANS--PE24 Adduct
H
H N
H H
N
I
O
SH
H
H
N
SO3
O
Cys residue of ETA mutant
IAEDANS
H
H N
O
N
S
O
N
H
H
SO3
AEDANS-labeled ETA mutant
+ HI
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
PE24
– Maximum Contact Model
eEF2
PE24
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Experimental Approach

Single cysteine residues introduced into PE24 at
21 defined sites and labeled with the fluorophore,
IAEDANS
A-519
G-525
S-515
O
NHCH2CH2NH C
G-549
Q-603
CH2 I
A-476
T-554
R-490
T-442
S-459
S-507
E-486
S-585 N-577
T-564
SO3H
S-410
Q-592
Q-428
S-449
S-408
Q-415
• fluorescence studies performed in the presence and absence of
eEF2
– acrylamide quenching
– fluorescence lifetime
– wavelength emission maximum
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Emission Max and Lifetime
1.5
3
1.0
  (lifetime) ns
1
0
0.0
-0.5
-1.0
408
410
415
428
442
449
459
476
486
490
507
515
519
525
549
554
564
577
585
592
603
-1
-1.5
Residue Num ber

0.5
No large shifts in  emission
maxima after eEF2 added
• 3 nm red shift for S449CAEDANS and S515C-AEDANS
• 1 nm blue shift for A519CAEDANS
408
410
415
428
442
449
459
476
486
490
507
515
519
525
549
554
564
577
585
592
603
  em max
2
Residue Number

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
9

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
F0
 K SV [Q]  1
Fi
K SV   0 k q
0.37  0.01
0.36  0.01
1.28  0.04
0.86  0.03
0.73  0.01
0.54  0.01
0.56  0.02
0.60  0.02
0.20  0.01
0.27  0.01
0.94  0.03
0.89  0.03
0.42  0.02
0.58  0.01
1.29  0.04
1.27  0.04
0.48  0.02
0.70  0.02
0.80  0.02
0.55  0.03
0.33  0.01
0.41  0.01
1.23  0.04
0.96  0.01
0.61  0.02
0.75  0.02
0.65  0.03
0.88  0.04
0.35  0.01
0.32  0.01
0.50  0.02
1.10  0.04
0.27  0.01
0.74  0.01
20 Q592C
0.90  0.02
1.42  0.02
0.59  0.01
0.79  0.01
21 Q603C
0.68  0.02
0.39  0.01
1
S408C
2
S410C
3
Q415C
4
Q428C
5
T442C
6
S449C
7
S459C
8
A476C
9
E486C
10 R490C
11 S507C
12 S515C
13 A519C
16 T554C
17 T564C
18 N577C
19 S585C
1.0
[Q]
-1 -1
1.03  0.02
1.29  0.01
Protein
15 G549C
KSV
9
kq x 10 M s
(+ eEF2)
14 G525C
F0/Fi
-1 -1
kq x 10 M s
(– eEF2)
28
Acrylamide Quenching
Protein
Adduct
kq(+ eEF 2) /
kq(– eEF 2)
S410C
S408C
T442C
T554C
E486C
S507C
S459C
S449C
A519C
T564C
R490C
G549C
Q592C
Q415C
Q603C
Q428C
A476C
S585C
N577C
S515C
G525C
3.53
2.79
2.77
2.74
2.69
2.44
2.26
2.25
2.04
1.85
1.82
1.79
1.79
1.75
1.75
1.60
1.54
1.54
1.48
1.34
1.28
4.0
*
3.0
*
*
*
*
*
2.5
* *
*
2.0
1.5
1.0
408
410
415
428
442
449
459
476
486
490
507
515
519
525
549
554
564
577
585
592
603
kq(-eEF2) / kq(+eEF2)
3.5
Residue Number
29
Model of PE24-eEF2 Complex

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
Jørgensen, et al., (2003) Nat. Struc. Biol. 10, 379-385 (eEF-2
structure); Li et al (1996) PNAS 93, 6902 (PE24 structure)
eEF2
Domain IV
a
b
Diphthamide
7
3
c
d
4
2
1
5
PE24
6
8
9
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FRET Experiments
R=Ro(E-1-1)1/6
E=1-FDA/FD
Ro=(K2JDAQDn-4)1/6 (9.79x103) Å
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Using 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
Donor
Fluorescence
Acceptor
Absorption
PE24AEDANS
eEF2Fluorescein
Wavelength ()
32
Labeling eEF2 with Fluorescein (Acceptor)
O
O
HO
O
O
 HI
C
OH
C
Fluorescein
O
NH
C
OH
Protein adduct
O
CH2
I
NH
..
HS
CH2
C
CH2
S
CH2
eEF2
eEF2
eEF2
eEF2--5-AF
1.0
0.8
Absorbance
HO
0.6
0.4
0.2
0.0
250
300
350
400
450
Wavelength (nm)
500
550
600
33
PE24-AEDANS Binding with eEF2-5AF

Created S585C mutant toxin (WT activity)
• labeled Cys at 585 with IAEDANS
Fractional Saturation (F/Fmax)
1.0
Dissociation constant (Kd)
0.8
– S585C-AEDANS
• 0.71 ± 0.08 mM
0.6
0.4
0.2
0.0
0
1000
2000
3000
4000
[eEF2-5AF], (nM)
(Armstrong et al. (2002) JBC 277:46669)
34
FRET Approach
T812C
T574C
Diphthamide
eEF2
PE24
35
Future Work

Determine kinetic mechanism for monoADPRTs
• 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
36
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