Transcript protws4 4761

The Role of the Proximal Tail in the
Large Steps of Myosin VI
Ron Rock
University of Chicago
Myosin II and F-Actin Architecture
Catalytic Domain
Coiled Coil
RLC
ELC
Myosin II:
Hexamer of
2 Heavy Chains &
4 Light Chains
F-Actin: Polymer of actin monomers
36 nm
Pointed End
Barbed End
The Myosin II Chemomechanical Cycle
Step Size and the Lever Arm
The light chain binding
domain is believed to
rotate upon binding to
actin
D
Result: Small
structural changes in
the catalytic domain
are amplified
Step Size Correlates to Lever Arm
Length for Myosin II
• Velocities in gliding filament assays
correlate
Uyeda et al. PNAS 93 4459 (1996)
• Step sizes correlate
Warshaw et al. JBC 275 37167 (2000)
Ruff et al. Nat. Str. Biol. 8 226 (2001)
Myosin Classes
Myosin Properties
Direction Processive
Myosin II
Barbed
Step
No
5 nm
Lever
Arm
Myosin V
Barbed
Yes
36 nm
Lever
Arm
Myosin VI
Pointed
Yes
30-36
nm
Total Internal Reflection Microscopy
Myosins V and VI are Processive
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V
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VI
A Hand-Over-Hand Model
• Myosin V and VI walk…
• … in a hand-over-hand manner …
• … using two catalytic heads
A Hand-Over-Hand Model
D
rate-limiting
T
P
Hand-Over-Hand Motility
Matthew L. Walker, Stan A. Burgess, James R. Sellers, Fei Wang,
John A. Hammer III, John Trinick & Peter J. Knight.
Nature, 405 , 804-807 (2000).
Myosin V Can Cross Actin Filaments
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Two Actin Tracks
Q ui ck Ti m e ™ an d a d ec om p r es so r a re ne
How Does Myosin V Cross Filaments?
Flexibility here?
Side View
Diffusive Search?
Top View
Optical Trap Design
Brightfield
Trap Steering
Dual Bead Force Clamp
Displacement (nm)
Myosin V Stepping in the Trap
480
400
320
240
160
80
5.45
5.5 5.55
Time [s]
0
0
1
2
3
4
Time (s)
2 mM ATP
5
6
Myosin VI Stepping in the Trap
VI
V
Myosin V and VI Takes Large Steps
100
F = 1.7 ± 0.2 pN
Count
80
60
VI
40
20
0
-40 -30 -20 -10 0 10 20 30 40 50 60 70
Displacement (nm)
50
Count
40
30
10
0
-40 -30 -20 -10 0 10 20 30 40 50 60 70
Displacement (nm)
• (VI) Large steps, much
larger than expected
from lever arm model
• (VI) Distribution very
broad (30 ± 12 nm)
F = 1.0 ± 0.2 pN
20
• Mean step is near the
actin helical repeat
V
• (VI) Many backsteps
(toward barbed end)
(-13 ± 8 nm)
Myosin V and VI Stepping Model
Coiled-coil unfolds
How does myosin VI take such large steps?
100
F = 1.7 ± 0.2 pN
Count
80
60
VI
40
20
0
-40 -30 -20 -10 0 10 20 30 40 50 60 70
Displacement (nm)
50
F = 1.0 ± 0.2 pN
Count
40
30
20
10
0
-40 -30 -20 -10 0 10 20 30 40 50 60 70
Displacement (nm)
V
The two heads of myosin VI can separate
27 ± 6 nm (SD)
The proximal tail is not predicted to be a
coiled-coil
Less than half of the processive stepsize is
generated by the working stroke
Similar to Myosin V: Veigel et al., Nat. Struct. Bio. 4 59 (2002)
The proximal tail does not act as a rigid
lever arm
N = 195
11.9 ± 1.2 nm (SE)
The proximal tail does not act as a rigid
lever arm
X
The proximal tail is exposed and sensitive
to proteolysis by V8 protease
Solid arrows indicate 97 kD band. Uncut Myosin VI is 146 kD. Actin:myosin
is at 6:1 mol ratio and nucleotides are at 2 mM unless indicated.
The proximal tail allows separation of the
heads to produce a large step
M6-2hepzip
V858 to S888 -> GCN4
19 ± 2 nm (SD)
The proximal tail allows separation of the
heads to produce a large step
Myosin VI and 2hepzip stepping model
Myosin VI stepping model
Myosin VI stepping model
80 AA => 28.8 nm (contour length) each
stiffness k = 0.3 pN/nm (WLC, Lp = 0.9
nm, constant over these ranges)
t k   / k  / 4 kBT /U0 exp(U0 / kBT )
First passage time under zero ext. load
(26 nm) is ~6 ms
Under 2 pN load, first passage time is 3 s
• Dock proximal tail along the
actin filament
• Alpha helical proximal tail
Acknowledgments
Protein production, EM,
kinetics
Bhagavathi Ramamurthy
Sara Beccafico
Carl Morris
Clara Franzini-Armstrong
H. Lee Sweeney
Optical Trapping,
proteolysis
Alex Dunn
Ben Spink
Bhadresh Rami
Jim Spudich
The Helen Hay Whitney Foundation
The Burroughs Wellcome Fund
Full-length myosin VI is a monomer
A form of motor regulation like Unc104?
EM of Myosin VI Decorated Actin Shows
Evidence of Left Handed Rotation
Pointed End
ADP
Rigor
Barbed End
Wells et al. Nature 401 505 (1999)
Load and the Diffusive Search
F = 1.7 pN
50 pN•nm =
200,000x slower
30 nm
F = 1.7 pN
0 pN•nm
30 nm
F = 1.7 pN
30 nm
25 pN•nm =
400x slower
ADP Release is the Rate Limiting Transition
2 mM ATP, 400 µM ADP
k0 = 6.4 s-1, k1 =161 s-1
ADP
10 µM ATP ( = Km)
k0 = 9 s-1, k1 =17 s-1
Single Fluorophore Detection
Nd:YAG 532 nm
HeNe 633 nm
Ar Ion 488 nm
Adapted from Tokunaga BBRC 235 47 (1997)