Combining isothermal titration calorimetry, fluorescence ... · Combining isothermal titration...
Transcript of Combining isothermal titration calorimetry, fluorescence ... · Combining isothermal titration...
Combining isothermal titration calorimetry, fluorescence anisotropy and kinetic approaches to decipher the molecular mechanisms of the
redox activation of the Yap1 transcription factor in S. cerevisiae
UMR 7365 IMOPA
Structural and Molecular
Enzymology
Nancy, France
Antoine Bersweiler
Alexandre Kriznik
Hortense Mazon
Dr B. D’Autréaux
Dr A. Delaunay-Moisan
Dr G. Belli
Dr M. Toledano
LSOC
CEA Saclay
France
Dr. S. Rahuel-Clermont
H2O2Reactive oxygen species = oxidant
ToxicCellular messenger
Alteration of biomolecular
function
Stress proteins ChaperonesEnzymes/transcription
factors activation
Normal physiological conditions
Oxidative stress
H2O2 local concentration
H2O2 as a signaling molecule
Stimuli UV Growth factor deprivation
p66shc H2O2
Stimuli Growth factors Cytokines
NOX
H2O2
H2O2 as a signaling molecule: issues
Specificity
Concentration tightly regulated by cellular antioxidant systems low in vivo concentrations
Signal Transduction ?
Molecular mechanisms that generate specificity in the transmission of oxidative
stress signals or redox signals ?
Yap1reduced
Yap1oxidized
H2O2
Delaunay et al., EMBO J 2000, Delaunay et al. Cell 2002Veal et al. JBC 2003
Yap1: redox activation of transcription factor by thiol peroxidase
H2O2
75 Kda
Yap1reduced
Yap1oxidized
Orp1
H2O2
Delaunay et al., EMBO J 2000, Delaunay et al. Cell 2002Veal et al. JBC 2003
= thiol peroxidase
Yap1: redox activation of transcription factor by thiol peroxidase
H2O2
H2O2
Ybp1
Orp1
79 kda18 kda
75 Kda
-Yap1S
598
S303
Orp1SH
SH
36
82
Orp1SOH
SH
36
82
H2O2
H2O
H2O
-Yap1SH
598
SH303
-Yap1S
598
SH303
Orp1HS
S36
82
Based on D’Autréaux et Toledano, Nature Reviews, 2007
Yap1: the redox relay 1st step of activation pathway
Relay of oxidative equivalents
Oxidative activation of Yap1 is sequential
Based on D’Autréaux et Toledano, Nature Reviews, 2007
-Yap1S
598
S303
Orp1SH
SH
36
82
Orp1SOH
SH
36
82
Orp1S
S
36
82
H2O
H2O2
H2O
H2O
-Yap1SH
598
SH303
Trx
-Yap1S
598
SH303
Orp1HS
S36
82
Yap1: Specificity?
Competition peroxidase/Yap1
Based on D’Autréaux et Toledano, Nature Reviews, 2007
-Yap1S
598
S303
Orp1SH
SH
36
82
Orp1SOH
SH
36
82
Orp1S
S
36
82
H2O
H2O2
H2O
H2O
-Yap1SH
598
SH303
Trx
Competition peroxidase/Yap1
-Yap1S
598
SH303
Orp1HS
S36
82
Yap1: Specificity?
Functional coupling betweenOrp1 and Yap1?
Direct recognition Orp1/Yap1?
Based on D’Autréaux et Toledano, Nature Reviews, 2007
-Yap1S
598
S303
Orp1SH
SH
36
82
Orp1SOH
SH
36
82
Orp1S
S
36
82
H2O
H2O2
H2O
H2O
-Yap1SH
598
SH303
Ybp1 ?
Trx
Competition peroxidase/Yap1
-Yap1S
598
SH303
Orp1HS
S36
82
Yap1: Specificity?
Functional coupling betweenOrp1 and Yap1?
Direct recognition Orp1/Yap1?Role of Ybp1 ?
Mechanism of Ybp1 action? Protein-protein interactions
Orp1SH
SH
36
82
-Yap1SH
598
SH303
Ybp1
Molecular recognition betweenthe redox relay partners?
E
polarizedligth
excitation
rapid rotation hν’ stronglydepolarized lightweak anisotropy
slow rotationhν’ weakly
depolarized lightstrong anisotropy
L
fluorescent probe: Alexa Fluor (AF) 488 5-SDP Ester
RL
absorption emission
Fluorescence anisotropy
Mechanism of Ybp1 action? Protein-protein interactions
Orp1SH
SH
36
82
-Yap1SH
598
SH303
Ybp1
Molecular recognition betweenthe redox relay partners?
Mechanism of Ybp1 action? Protein-protein interactions
Orp1SH
SH
36
82
-Yap1SH
598
SH303
Ybp1
Molecular recognition betweenthe redox relay partners?
Ybp1KD = 1,3 μM
Ybp1+-HS
HS
598
303
Yap1 -
598
303
Yap1SH
SH
Yap1·Ybp1 1·1
stoichiometry = 1:1
%
m/z
Ybp1
MW = 79780 Da
MW = 154296 Da
Ybp1
-598
303
Yap1SH
SH
Ybp1
-Yap1SH
SH
Collaboration S. Cianférani, Strasbourg
Native mass spectrometry
Ybp1 recruits Orp1 and Yap1 into a ternary complex
Orp1SH
SH
36
82
-Yap1SH
598
SH303
Ybp1
-Yap1SH
598
SH303
Ybp1
ITC titration of Yap1, Ybp1 and Yap1-Ybp1 by Orp1
kcal
mol
-1 O
rp1
inje
cted
time (min)
μca
l/sec
molar ratio
Orp1 and Yap1 do not interact
-Yap1HS
HSOrp1
SH
SH+
Orp1SH
SH
kcal
mo
l-1 O
rp1
inje
cted
time (min)
μca
l/sec
molar ratio
μca
l/sec
time (min)
kcal
mo
l-1 O
rp1
inje
cted
molar ratio
-Yap1HS
HSOrp1
SH
SH+ Orp1
SH
SHYbp1 +
Ybp1
KD = 0,8 μMN = 1
Ybp1 and Orp1 form a 1·1 complex
μca
l/sec
kcal
mol
-1 O
rp1
inje
cted
time (min)
molar ratio
Orp1 interacts with Ybp1·Yap1
Two hypotheses
μca
l/sec
kcal
mol
-1 O
rp1
inje
cted
time (min)
molar ratio
Orp1 interacts with Ybp1·Yap1
Two hypotheses
KD = 0,7 μMN = 1
Orp1 does not interact with Yap1Orp1 interacts with Ybp1
Orp1SH
SH
36
82
-Yap1SH
598
SH303
Ybp1
-Yap1SH
598
SH303
Ybp1
X
Ybp1 recruits Orp1 and Yap1 into a ternary complex
-Yap1S
598
S303
Orp1SOH
SH
36
82
H2O
-Yap1SH
598
SH303
-Yap1S
598
SH303
Orp1HS
S36
82
Ybp1 ?
kYap
H2O2
Orp1SH
SH
36
82
X
X
Rate of the first step of Yap1 activation pathway
H2O
X
Orp1S
S
36
82
Role of ternary complex in Yap1 oxidation mechanism?
Orp1SOH
SH
36
82
H2O
-Yap1SH
598
SH303
-Yap1S
598
SH303
Orp1HS
S36
82
Ybp1 ?
kYap
H2O2
Orp1SH
SH
36
82
XH2O
Reconstitution of first step of activation pathway in vitro
ms to min incubations Reverse phase chromatographic
analysis (C8)
Elution time (min)
abso
rban
ce (2
15 n
m)
Orp1SH
SH
-Yap1HS
HS
→Ybp1
kYap determination
Reactiontime (s)
S
HSOrp1Orp1
SH
S
S
-Yap1S
HSOrp1
SH
Orp1SH
SH
-Yap1HS
HS
Orp1SH
SNH
Elution time (min)
abso
rban
ce (2
15 n
m)
Orp1SH
SNH
→Ybp1
kYap determination
Reactiontime (s)
Elution time (min)
abso
rban
ce (2
15 n
m)
S
-Yap1S
HSOrp1
SH
Orp1SH
SH
-Yap1HS
HS
Orp1SH
SNH
S
HSOrp1Orp1
SH
S
→Ybp1
kYap determination
Reactiontime (s)
Elution time (min)
abso
rban
ce (2
15 n
m)
S
-Yap1S
HSOrp1
SH
Orp1SH
SH
-Yap1HS
HS
Orp1SH
SNH
S
HSOrp1Orp1
SH
S
→Ybp1
kYap determination
Reactiontime (s)
Elution time (min)
abso
rban
ce (2
15 n
m)
S
-Yap1S
HSOrp1
SH
Orp1SH
SH
-Yap1HS
HS
Orp1SH
SNH
S
HSOrp1Orp1
SH
S
→Ybp1
kYap determination
Reactiontime (s)
Elution time (min)
abso
rban
ce (2
15 n
m)
observed rate constant = 0.3 s-1
observed rate constant = 2.6 s-1
S
-Yap1S
HSOrp1
SH
Orp1SH
SH
-Yap1HS
HS
Orp1SH
SNH
S
HSOrp1Orp1
SH
S
Ybp1
Ybp1
fraction of total
time (s)
Ybp1
Ybp1
S
-Yap1S
HSOrp1
SH
Ybp1 accelerates the reaction Orp1-SOH + Yap1
Reactiontime (s)
Elution time (min)
abso
rban
ce (2
15 n
m)
observed rate constant = 0.3 s-1
observed rate constant = 2.6 s-1
Wild type Orp1 = 5 s-1
S
-Yap1S
HSOrp1
SH
Orp1SH
SH
-Yap1HS
HS
Ybp1
Ybp1
fraction of total
time (s)
Ybp1
S
-Yap1S
HSOrp1
SH
Ybp1 accelerates the reaction Orp1-SOH + Yap1
Reactiontime (s)
Orp1S
S
kYap
Ybp1
Orp1
In vivo Orp1 concentration = 0,5 µMExperiments @ 50 µM
Real effect of complex formation = 600 fold
Role of ternary complex in Yap1 oxidation mechanism?
kYap = Cste suggests that the reaction occurs within the ternary complex
Simulation of kYap = f(Orp1) based on KD of 0,7 µM
In the absence of Ybp1 the reaction is bimolecular
Orp1SH
SH
36
82
Orp1SOH
SH
36
82
Orp1S
S
36
82
H2O
H2O2
H2O
kSS = 500 s-1
Intramolecular disulfide formation in Orp1 is very fast
Ma et al. JBC 2007
Orp1SH
SH
36
82
Orp1SOH
SH
36
82
Orp1S
S
36
82
H2O
H2O2
H2O
kSS
Ybp1
Trx
Does Ybp1+Yap1 inhibit formation?
Within
kss decreased from 500 to 6 s-1
Reduced competition with Yap1
Reaction between Orp1 and Yap1 is possible
time (min)
kcal
mol
-1 O
rp1
inje
cted
μca
l/sec
time (min)
kcal
mol
-1 O
rp1
inje
cted
μca
l/sec
molar ratio
The disulfide form of Orp1 doesn’t interact with Ybp1 and Yap1·Ybp1
-Yap1HS
HS+ Ybp1 + +
molar ratio
μca
l/sec
kcal
mol
-1 O
rp1
inje
cted
time (min)
molar ratio
Orp1S
SOrp1
S
SOrp1
S
SYbp1
-Yap1SH
SH
time (min)
kcal
mol
-1 O
rp1
inje
cted
μca
l/sec
time (min)
kcal
mol
-1 O
rp1
inje
cted
μca
l/sec
molar ratio
The disulfide form of Orp1 doesn’t interact with Ybp1 and Yap1·Ybp1
-Yap1HS
HS+ Ybp1 + +
molar ratio
μca
l/sec
kcal
mol
-1 O
rp1
inje
cted
time (min)
molar ratio
Orp1S
SOrp1
S
SOrp1
S
SYbp1
-Yap1SH
SH
-Yap1SH
598
SH303
Orp1SH
SH
36
82Ybp1
-Yap1SH
598
SH303
Ybp1 recruits Orp1 (ITC)
Bersweiler et al., in revision
Yap1 activation mechanism
Orp1SH
SH
36
82Ybp1
-Yap1SH
598
SH303
Ybp1
-Yap1SH
598
SH303
Orp1SOH
SH
36
82
-Yap1SH
598
SH303
Ybp1 kYap1
Ybp1 recruits Orp1 (ITC)Orp1 disulfide formation is decreased within the ternary complex
Bersweiler et al., in revision
Yap1 activation mechanism
Orp1SH
SH
36
82Ybp1
-Yap1SH
598
SH303
Ybp1
-Yap1SH
598
SH303
Orp1SOH
SH
36
82
-Yap1SH
598
SH303
Ybp1 kYap1
Ybp1 recruits Orp1 (ITC)Orp1 disulfide formation is decreased within the ternary complex
Bersweiler et al., in preparation
Yap1 activation mechanism
Orp1SH
SH
36
82Ybp1
-Yap1SH
598
SH303
Ybp1
Orp1SOH
SH
36
82
-Yap1SH
598
SH303
Ybp1 kYap1
kSS
Ybp1 recruits Orp1 (ITC)Orp1 disulfide formation is decreased within the ternary complex
Bersweiler et al., in preparation
Yap1 activation mechanism
Orp1SH
SH
36
82Ybp1
-Yap1SH
598
SH303
Ybp1
-Yap1SH
598
SH303
signal
Sensor/receptor
modified sensor
sensing
transducer
transduction
effector
Case of redox signaling
Oxidant
sensing
transducer
transduction
effector
Signaling pathway
Cellular signaling pathways
proteinS-
proteinSox
Molecular recognition
Oxidation
Molecular recognition
In vitro reconstitution of the redox relayRecombinant purified proteins
Collaboration M. Toledano
Ybp1
High H2O2
Low H2O2
Conclusion: requirement of both Orp1 and Ybp1 in efficient and sensitive Yap1 oxidation by H2O2
H2O2
proliferation growth arrest apoptose10-8 10-6 10-4 local
concentration (M)
proteinSox
cellular response
effector
proteinS-
sensorSox
sensorS-
H2O2
relay
sensing
lower reactivity in most cases
high reactivity with H2O2
OxyR
signaling
Prx2/Stat3
Sobotta et al., Nature Chemical Biology, 2015
oxidative stress
Direct/indirect sensing of the H2O2 signal
sensorS-
= Enzyme family Thiol peroxidase
Table 1. Binding and thermodynamic parameters of Orp1 binding to Ybp1 and the Yap1SSS SSS·Ybp1
complex deduced from ITC titrationsa
Dissociation constant (µM)
Stoichiometry ΔH cal mol-1
-TΔS cal mol-1
Ybp1 0.8 ± 0.1 0.8 ± 0.1 - 4890 ± 400 - 3430
Yap1SSS SSS·Ybp1 0.7 ± 0.2 0.8 ± 0.1 - 4210 ± 240 - 4235 a Parameters deduced from the analysis of binding isotherms shown Fig. 3C
Orp1SH
SH
36
82
Orp1SOH
SH
36
82
Orp1S
S
36
82
H2O
H2O2
H2O
kSS
0 1 2 3 4 51.5
2.0
2.5
3.0
3.5
H2O
2 5 to 800 µM
Flu
ore
scen
ce in
ten
sity
Time (s)
0.00 0.01 0.02 0.03 0.04 0.051.5
2.0
2.5
3.0
3.5B
Flu
ore
scen
ce in
ten
sity
Time (s)
A
H2O
2 1 to 20 mM
kSOH = 1.5 105 M-1s-1
kSS = 500 s-1
Intramolecular disulfide formation in Orp1 is very fast
Ybp is required for the reaction WT Orp1-SOH + Yap1
Orp1 model
Ma et al. JBC 2007
Non reducing SDS-PAGE