chemomechanical coupling in myosin ii : experiment vs....
TRANSCRIPT
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Chemomechanical Coupling in Myosin II :
Experiment vs. Theory
Clive R. BagshawDepartment of Biochemistryhttp://www.le.ac.uk/biochem
Physics of Moleculer Machines Les Houches 2006
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Topics: Myosin II
• States identified from crystal structures• Switch movements probed by
tryptophan fluorescence• Domain movements probes by other
fluorophores• Coupling of movements to Pi release
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Crossbridge cycle based on crystal structures
Animation by Ken Holmes
Model based on two (now three) crystal structures of myosin and em of decorated filaments
Does the neck really swing?
How is tail swing reversed by actin?
Is the lever arm swing the only mechanism contributing to sliding?
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Sw2
P-loopSw1
From Geeves & Holmes 2005
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ATP
Sw1
Sw2
ACTIN
A.M (actomyosin)
Cleft closed, Switch 1 and Switch 2 open
P-loopcleft
lever arm
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ATP
Sw1
Sw2
M†.ATP
Switch 1 closes, Cleft opens, actin dissociates
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Sw1
ADP.Pi
M*.ADP.Pi
Sw2
Switch 2 closes, ATP hydrolysis occurs
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ADP.Pi
Sw2
Sw1ACTIN
A.M*.ADP.Pi
Actin binds, cleft closes, Switch 1 opens
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ADP
Sw1
Sw2
ACTIN
A.M*.ADP
Pi released and Switch 2 opens = stroke
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But ....
Holmes, Schröder, Sweeney & Houdusse (2004) Phil. Trans. R. Soc. B 359, 1819-1828
They proposed that relay helix could unkink without switch 2 having to open. This was brought about by the twisting of the core 7-strand β-sheet which moved F652 (the proposed cause of the relay helix kinking)
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Sw1
ADP.PiA
CTIN
A.M*.ADP.Pi (weakly bound)
Sw2
Cleft closes, Switch 1 opens
F652
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Sw1
Sw2
ACTIN
A.M*.ADP.Pi
The β−sheet twist model
Switch 2 remains closed during lever arm swing
ADP.Pi
F652
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Sw1 O C C O O
Sw2 O O C C O
A.M A.M.T A.M.D.P A.M.D.P A.M.D
Stroke = Sw2 opening +Pi release
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Sw1 O C C O O
Sw2 O O C C O
A.M A.M.T A.M.D.P A.M.D.P A.M.D.P
Stroke = Sw2 opening before Pi release
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Sw1 O C C O O
Sw2 O O C C O
A.M A.M.T A.M.D.P A.M.D.P A.M.D
Stroke = relay helix unkinking due to β sheet twist
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1 = Houdusse
2 = Holmes (but not quite the same closed)
3 = Kull (P loop as ref)
Author myosin state 50K actin cleft
switch 1 switch 2 β sheet lever arm
jargon
Rayment Dicty 1MMD
ADP.BeFx open closed open straight down post rigor
Dicty1MND
Courreux myosinV 10E9
apo closed open different1
closed2
open3
twist down rigor-like
closed straight up pre power
Rayment ADP.AlF4 open closed
Key crystal sates
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1 = Houdusse
2 = Holmes
3 = Kull (P loop as ref)
Author myosin state 50K actin cleft
cleft base
switch 1 switch 2 β sheet lever arm
jargon
Rayment skeletal 1mys
apo βS04 open open closed open down post rigor
Rayment Dicty 1MMD
ADP.BeFx open open closed open down post rigor
Holmes/Kullunpublished
Dicty
1MNDHoudusse Scallop
B7TADP open closed open more
downCourreux myosinV
10E9apo closed open open different1
closed2
twist down rigor-like
Reubold Dicty 1Q5G
apo intermediate
open open3
closed2
twist down on the way to rigor
pre-power
Rayment ADP.AlF4 open closed closed closed up pre power stroke
closed closed upDicty ADP.BeFx open closed
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ATP
W239 •
Sw2
Fluorescence Probes of domain movement
• W501
BFPGFP
Pyrene
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Computational Chemistry
G. Li, Q. Cui, (2004) Mechanochemical coupling in myosin. A theoretical analysis of ATP hydrolysis with molecular dynamics and combined QM/MM reaction path calculations, J. Phys. Chem. B 108,3342-3357.
G. Li, Q. Cui, (2004) Analysis of functional motions in "Brownian molecular machines" with an efficient block normal mode approach. Myosin-II and Ca2+-ATPase Biophys. J., 86, 743-763
Schwarzl, S. M., J. C. Smith, and Fischer, S. (2006). "Insights into theChemomechanical Coupling of the Myosin Motor from Simulation of Its ATP Hydrolysis Mechanism." Biochemistry 45(18): 5830-5847
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Nucleotide binding to W501+( saturating [nucleotide] at 20oC )
0.32
0.34
0.36
0.38
0 0.05 0.1 0.15 0.2 0.25 0.3
Fluo
resc
ence
(vol
ts)
Time (s)
ATP
ATPγS
ADP
M*.ADP.Pi
M†.ATPγS ↔ M*.ATPγS
M†.ADP
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ATP Hydrolysis Step has been Resolved into Two Steps
M†.ATP M*.ATP M*.ADP.PiOpen state Closed state Closed state
K3a K3b
Temp
Pressure
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Conclusions from W501 fluorescence
• Model independent– Relay helix and/or converter domain moves in response to
ATP binding, but hydrolysis is required to make equilibrium favorable
– Reaction is freely reversible, even when hydrolysis occurs
– Actin has no direct effect on this reaction
• Model dependent– W501 senses switch 2 and lever arm movement (consistent
with crystal structures)
– Actin influences lever arm movement through Switch 1
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Key Question
What is the difference between the lever arm swing in the absence and presence of actin?
Depends on Switch 1 position ?
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Coupling between actinbinding and switch 1
• Is there a 1:1 coupling between cleft closure and switch 1 opening?
• Answer: not simple as there multiple states of switch 1 conformation
• ….. but there is evidence of some reciprocity between 50k cleft closure and switch 1 opening.
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Conclusions from W239+
• Apo and +MgADP states comprise a mixture of conformers
• MgATP and actin lock the W239 in a “single”state with respect to W239 with low and high fluorescence respectively.
• This is consistent with structural ideas about switch 1, but there are more than two states
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Comparison of W501 and W239
W501
MgADP and apo single states
MgATP and MgATPγS two states
Little sensitivity to actinbinding
W239
MgADP two states
MgATP and MgATPγS single states
Sensitive to actin binding
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ATP
ATP
A B
C D
510 nm
FRET
380 nm
510 nm
380 nm
ESPT
5 nm
Effect of intramolecularGFP-BFP association
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Conclusions from FRET experiments
± 10 Å
(κ2 )
- 1.5 Å0.58
0.61
Apo YFP-M-CFP
+ ATP
Zeng et al 2006
± 13 Å
(κ2 )
+ 1.5 Å0.70
0.66
Apo GFP-M-BFP
+ ATP
Zeng et al 2006
± 1.4 Å(S.D.)
+ 15Å0.439
0.082
Apo GFP-M-BFP
+ ATP
Suzuki et alNature 1998
UncertaintyDistance change
FRET efficiency
ConstructAuthor