Supplementary material includes :
1- This word document containing the figure legend and material and methods concerning
the three figures proposed for supplementary materials
2- Supplementary Figure 1
3- Supplementary Figure 2
4- Supplementary Figure 3
SUPPLEMENTARY MATERIALS
Supplementary Figure 1. The actin-myosin ATPase cycle. Shown are the states in the
actomyosin–ATPase cycle that have been seen in either high resolution structures of myosin
(near-rigor and transition state), or in low resolution actomyosin structures derived from cryoEM
(strongly bound ADP state and rigor). The cycle leaves out a number of intermediate states that
are known to exist from kinetic studies. The lever arm movement that produces force occurs
primarily upon binding to actin and release of the inorganic phosphate. In some myosins, an
additional lever arm rotation is associated with ADP release. Repriming of the lever arm occurs
during the isomerization from the near-rigor to the transition state, which allows hydrolysis of
ATP.
Supplementary Figure 2. Rates of binding of the MV-1IQ and SkS1 to pyrene actin filaments
and the dependence on actin concentration. The rates of MV-1IQ and SkS1 binding to pyrene
actin in the absence of nucleotide are plotted as a function of pyrene actin concentration. Linear
fits of the rates (kobs) of MV-1IQ binding to pyrene actin at 25 C (closed squares) and 30 C
(open squares). Hyperbolic fits of the rates (kobs) of SkS1 binding to pyrene actin at 25 C (closed
circles) and 30 C (open circles).
Methods:
2
Transient Kinetic Experiments. Papain digested skeletal muscle myosin subfragment 1 (SkS1)
was prepared as described by Margossian and Lowey (Margossian SS, Lowey S. Preparation of
myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 85:55-71 (1982)).
All transient kinetic experiments were performed with an Applied Photophysics (Surrey, UK)
stopped-flow model SX.18MV with a dead-time of 1.2 ms. Pyrene fluorescence was excited at
365 nm and the emitted fluorescence was measured using a 400 nm long pass filter (Oriel Corp.,
Stratford, CT).
Nonlinear least-squares fitting of the data was done with software provided by the
instrument or with Kaleidagraph (Synergy Software, Reading, PA). Uncertainties reported are
standard error of the fits unless stated otherwise. All transient kinetic experiments with
SkS1were performed in 10/10 buffer (10 mM Imidazole pH 7.0, 10 mM KCl, 1 mM MgCl 2, 1
mM EGTA, 1 mM DTT) and experiments with MV 1IQ were conducted in KMg50 buffer (10
mM Imidazole pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT).
Kinetic Modeling. The following kinetic scheme of Taylor (Taylor, E.W. Kinetic studies on the
association and dissociation of myosin subfragment 1 and actin. J. Biol. Chem. 266, 294-302
(1991)) was used to model the interaction between skeletal myosin II and pyrene actin filaments,
where A represents actin, M represents myosin, and an asterisk next to actin indicates the pyrene
actin fluorescence is unquenched (A*).
Scheme 1
K1
↔
k+2
A* + M
A*.M ↔
A.M
k-2
The initial collision complex between myosin and actin is thought to be a rapid equilibrium (K1)
process that does not quench pyrene actin fluorescence, and is followed by an isomerization step
(K2) that results in a strongly bound complex in which the pyrene fluorescence is quenched.
2
3
The rate of myosin binding to actin filaments was determined by mixing myosin with 10-fold
excess pyrene-actin and measuring the rate of pyrene fluorescence quenching. The rates of
binding to actin were plotted as a function of actin concentration and fit to a hyperbola or linear
regression. The initial rate was modeled to be K1k+2, the maximum rate k+2, and the actin
concentration at which there is half-maximal saturation was 1/K1.
Supplementary Figure 3. Overall view of the myosin V nucleotide-free structure. The
myosin V structure is compared to two weak binding states of the scallop myosin II (truncated
after the first IQ motif) in either the pre-power stroke state (transition state, PDB code 1DFL)
with the lever arm up or the near-rigor state (PDB code 1DFK) with the lever arm down. The
structures are oriented so that the lower 50kDa subdomains are superimposed and the arrows
indicate the direction of the actin filament axis when myosin binds according to docking in cryoEM maps. Note the position of the converter (green) and lever arm (cyan helix and pink essential
light chain) which are similar between the near-rigor and nucleotide-free (myosin V) states but
quite different between the transition and nucleotide-free states. The swing of the lever arm
between these two states would lead to force production, according to the swinging lever arm
hypothesis. The subdomains are colored as follows: upper 50 kDa is blue, the lower 50 kDa is
light gray, the N-terminal is dark gray, and the converter is green. Additional elements that are
highlighted by color include: the strut (between the upper and lower 50 kDa subdomains) in
fuchsia, switch II in orange, switch I in purple, P loop in light green, relay in yellow, SH1 helix
in red, and the lever arm helix in cyan. The essential light chain is shown in pink. The
conformation adopted by the essential light chain in its interaction with its IQ motif in the
myosin V lever arm is the same as found in the smooth muscle myosin II structure (PDB code :
1BR1).
3