 |
INTRODUCTION |
Myosin II, the major contractile protein of muscle, consists of
two globular catalytic domains, referred to as S1 subfragments, attached to a long coiled-coil rod. Insights into the mechanisms of
contraction were provided by x-ray crystal structures of individual S1
fragments from several myosin isoforms (1-3). From these studies it is
now apparent that each S1 can be divided into two subdomains; that is,
a globular motor domain that contains separate actin and nucleotide
binding sites and an unusual ~8.5-nm
-helical segment, the
so-called light chain binding domain, which connects the motor domain
to the coiled-coil rod. ATP hydrolysis at the catalytic domain is
thought to result in an angular rotation of the light chain binding
domain that corresponds to the force-generating "power stroke" of
the catalytic cycle of the enzyme (4).
Despite recent progress toward understanding the molecular basis of
force generation, significant questions regarding the structure and
function of myosin remain. In particular, since a high resolution
structure for a two-headed myosin molecule is not yet available,
comparatively little is known regarding potential head-head
interactions or the arrangement at the S1/S2 junction, i.e.
the region where each S1 domain joins the proximal (S2) section of the
myosin rod. In smooth and invertebrate myosins, activation of ATPase
activity is dependent on events occurring at this junction, such as
Ca2+ binding or phosphorylation of the regulatory light
chains, accessory proteins that bind the light chain binding domain
near the S1/S2 junction. For these regulated myosins, activation
appears to involve a significant conformational change that encompasses
both head-head and head-tail interactions (5). By contrast, activation
of unregulated myosins such as those of skeletal and cardiac muscle occurs primarily through events related to Ca2+ binding to
the thin filament. Nonetheless, characteristics of the S1/S2 junction
such as flexibility and angle of S1 attachment are still expected to
have significant effects on the magnitude and rate of force generation.
Consistent with this, regulatory light chain phosphorylation
potentiates force and the rate of force development in skeletal muscle
fibers (6).
Steric constraints or cooperative interactions between heads may also
affect motor function in unregulated myosins. For instance, steric
hindrance between the heads may result in nonequivalent actin binding
affinities, potentially leading to cooperative binding (7, 8).
Similarly, cooperativity was implied by single molecule force and
displacement measurements that indicated both heads are necessary for
maximum force generation by myosin (9). However, because spectroscopic
(10) and electron micrograph studies (11, 12) suggest the S1/S2
junction is quite flexible, a structural basis for head-head
interactions has been difficult to resolve.
In this paper, we present low resolution structural data on the overall
conformation of myosin in solution. Small-angle x-ray and neutron
scattering experiments were performed on heavy meromyosin (HMM),1 a soluble proteolytic
fragment of myosin. Neutron scattering measurements were used to obtain
structural information regarding the longest particle dimensions of HMM
that were necessary to accurately describe and model the molecule.
X-ray scattering measurements were used for comparison to confirm
overall HMM configuration as well as to check for monodispersity in
solution and assure there were no concentration-dependent
interparticle interference effects that would bias the modeling.
Comparisons of the neutron scattering intensity profiles with simulated
spectra generated from an existing model of the head-tail junction of
myosin (13) indicated that the model may not accurately represent the
structure of HMM in solution. Instead, experimental small- angle
scattering intensity profiles were reproduced by simple models in which
the S1 heads were joined asymmetrically to a coiled-coil rod.
 |
MATERIALS AND METHODS |
Sample Preparation--
Myosin was purified from rabbit back and
leg fast twitch skeletal muscle according to Wagner and Giniger (14).
HMM was prepared by limited
-chymotryptic digestion of myosin
according to Weeds and Pope (15). To minimize degradation of regulatory
light chain, myosin was digested in the presence of 2 mM
MgCl2 at a myosin:chymotrypsin ratio of 400:1 for 6 min at
22 °C. After precipitation of rod fragments and undigested myosin in
1 mM EDTA and 1 mM dithiothreitol, HMM in the
supernatant was dialyzed against 50 mM Tris-Cl, pH 7.2, and
further fractionated by fast protein liquid chromatography over a
Q-Sepharose ion exchange column. Peak fractions were pooled, mixed with
an equal volume of glycerol, and stored at
20 °C before use
(within 14 days) in scattering experiments.
For x-ray scattering measurements, purified HMM was dialyzed
extensively against experimental buffers containing 150 mM
KCl, 20 mM imidazole, pH 7.0, 1 mM EDTA, and 1 mM dithiothreitol. Solutions of similar composition were
used for neutron scattering experiments, except that D2O
was used in place of H2O to increase scattering signal,
which depends upon the scattering density difference between the
protein and solution (16). HMM scattering intensity profiles in
H2O and D2O solutions were comparable,
suggesting that HMM shape was not affected by the presence of
D2O. For solutions containing divalent ions, 2 mM MgCl2 or 2 mM CaCl2
was added to achieve ~1 mM free Mg2+ or
Ca2+. After dialysis, HMM samples were clarified by
ultracentrifugation (150,000 × g, 45 min) and
concentrated (3-30 mg/ml) using Centricon concentrators (Amicon).
Concentration of HMM was estimated assuming 350 kDa for molecular mass
and E280 nm = 6.47 cm
1 (15).
Sedimentation Velocity--
Sedimentation velocity values for
HMM were determined by sucrose density gradient ultracentrifugation as
described (17). Thyroglobulin, catalase, aldolase, and albumin
(Amersham Biosciences) were used as standards. 100 µg HMM or standard
was layered onto a linear 5-20% (w/v) sucrose gradient in buffer
containing 150 mM KCl, 20 mM imidazole, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol, and 2 mM MgCl2. Samples were centrifuged at 50,000 rpm for 90 min at 10 °C in a 65.1 VTi rotor. 0.5-ml fractions were
collected, sucrose concentrations were measured, and proteins were
analyzed by SDS-PAGE.
Stokes Radius--
The Stokes radius of HMM was determined by
gel filtration chromatography using a Superose 6 fast protein liquid
chromatography column and protein standards (albumin, catalase,
ferritin, thyroglobulin, and laminin). HMM and standards were
chromatographed in a solution containing 150 mM KCl, 20 mM imidazole, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol, and 2 mM
MgCl2.
Molecular weight was determined using the equation (18),
|
(Eq. 1)
|
where
= 0.010019 g/s·cm, N = 6.02 × 1023, Rs represents the
Stokes radius (cm), s represents the sedimentation
coefficient (Svedberg),
represents partial specific volume (g/ml),
and
20,w = 0.99823 g/ml.
X-ray Scattering Measurements--
Small-angle x-ray scattering
experiments were performed on the 2-m instrument at Los Alamos National
Laboratory (19). The instrument uses a sealed x-ray tube to produce
Cu-K
x-rays (1.54 A, 8.5 keV) focused to a vertical line
at the plane of a one-dimensional position-sensitive detector. Sample
and buffer data were collected to produce intensity profiles
I(q) versus q
(q = 4
(sin
)/
,
= wavelength, 2
= scattering angle) by following published procedures (19). The
q range used for data analysis was 0.01-0.15
Å
1. Data collection times were typically 1-4 h,
depending on the sample concentration, and the sample cell was
maintained at a constant temperature (7 ± 1 °C) over the
course of an experiment. To assess concentration dependence of
scattering, scattering data were collected on a dilution series for
each HMM protein sample and extrapolated at infinite dilution.
Molecular weight, Mr, was calculated by
comparing the forward scattering (I0) at
infinite dilution to that of a standard of known molecular weight
according to the following equation.
|
(Eq. 2)
|
Lysozyme (Mr = 14.2 kDa, Sigma) (20) was
used as the protein standard.
Neutron Scattering Measurements--
Neutron scattering data
were collected on the NG3 30-m SANS instrument at the National
Institute of Standards and Technology in Gaithersburg, MD. Data were
reduced according to published methods (21) to correct for detector
sensitivity and sample background. The neutron wavelength was set to
5.5 Å with a wavelength spread 
/
of 0.35 to maximize neutron
flux. Sample-to-detector distances of 13.1 and 1.7 m were used to
cover the appropriate q-range (0.003-0.025 and 0.021-0.27
Å
1, respectively). Intensity profiles for samples and
corresponding buffers were collected at both detector distances to
correct for background. HMM samples (8-10 mg/ml) and buffers were
maintained at 10 °C throughout data collection. Typical data
collection times were 4 h for the 13.1-m sample-to-detector
distance and 30 min for the 1.7-m distance. The data from the two
detector distances were merged using procedures included with the
National Institute of Standards and Technology data reduction software.
The higher contrast and lower measured minimum q value for
the neutron scattering experiments meant that these data have
significantly greater statistical precision and are able to more
accurately determine the scale parameters of the large HMM molecule.
Therefore, neutron scattering data were used for all model comparisons.
Small-angle Scattering Data Analysis--
The small-angle x-ray
or neutron scattering from a homogeneous solution of monodisperse
particles can be written as,
|
(Eq. 3)
|
where
(r) is the scattering length density
of the scattering particle and
s is the scattering length
density of the solvent. q is the momentum transfer, having
the magnitude given above. The integration over the particle volume is
rotationally averaged, and the experiment measures the time and
ensemble average for all particles in solution.
In addition to traditional Guinier analysis (22) of the data for the
radius of gyration, Rg, the probable distribution of
vector lengths within the scattering object,
P(r), can be determined from the scattering
intensity profile. I(q) and
P(r) are related by the Fourier transform in
Equation 4.
|
(Eq. 4)
|
The 0th and second moments of P(r) gives
the forward scatter (I0) and
Rg values for the scattering object typically with
greater precision than Guinier analysis because the calculations utilize the information in the entire measured scattering profile. The
indirect Fourier transform algorithm implemented in the program GNOM
(23, 24) was used to determine P(r) from the
measured intensity (25, 26). A slit smearing correction was applied to
the small-angle x-ray scattering data to correct for the instrument geometry. No correction for smearing was required for the neutron scattering data because the dimensions of the beam are adequately approximated as a point source for these experiments.
Computer Modeling--
To compare experimental scattering
spectra with a published model of HMM structure (13), simulated
scattering intensity profiles and P(r) functions
were generated from the atomic coordinates of the model using software
(PR_PDB) developed at Los Alamos National Laboratory (27). A second
program developed at Los Alamos National Laboratory, called XTAL_STR
(28), was used to produce composite models that fit the scattering
intensity profiles. The known structures can either be atomic
coordinates, such as crystal structure coordinates or coordinates of
points randomly distributed within the volume of a shape such as an
ellipsoid. Composite model structures are made by randomly positioning
and orienting the known structures with respect to one another. The
program assumes that the density of each component structure is uniform
to calculate P(r) from the coordinates of the
substructures. P(r) is transformed into I(q) through the transform defined in Equation 4.
The quality of the fit of the model intensity profile to the
experimental intensity profile is measured using F, a
modification of the reduced
-squared parameter, as defined in
Equation 5.
|
(Eq. 5)
|
Npts is the number of points in the data set,
I(q) and Im(q) are
the experimental and model intensity values, respectively, and
(q) is the experimental uncertainty of
I(q). The set of known structures used for the
modeling were generated from the Offer and Knight HMM model (13). The
original model was broken into three sections, the two S1 heads and the
coiled-coil tail. To obtain optimized models using the small-angle
scattering data, XTAL_STR was used such that the subunits were
constrained to remain in contact at fixed points. Model structures were
produced by randomly rotating each substructure around the connection
points. Thus, there were six degrees of freedom in the modeling done by XTAL_STR (two hinges with three Euler angles each). The published model
has a truncated coiled-coil tail; therefore additional models with
longer tails ranging from the length of the published structure (~180
Å) up to 1500 Å were tested to find the length that best fit the
data. The tail length used for the final modeling was ~380 Å. Each
XTAL_STR run tested in excess of 300,000 model iterations, and a single
best fit model was selected from each run. Three independent runs of
XTAL_STR were performed to determine the reproducibility of the modeling.
 |
RESULTS |
Purified HMM--
Fig. 1 shows
physical characteristics of purified HMM. In contrast to HMM produced
by tryptic digestion, HMM produced by mild
-chymotryptic digestion
is relatively homogenous (15). SDS-PAGE analysis (Fig. 1,
inset) showed that the HMM heavy chain migrated as a high
molecular mass band at ~140 kDa. All three light chain peptides were
also evident as bands at ~21 (LC1), 19 (regulatory
light chain), and 16.5 kDa (LC3). The sedimentation coefficient (Fig. 1A) and Stokes radius (Fig. 1B)
of HMM were determined by sucrose gradient ultracentrifugation and gel
filtration chromatography, respectively. A calculated value of 371 kDa
for the molecular mass of HMM was obtained using sedimentation
coefficient and Stokes radius values (see "Materials and Methods").
The value was similar to published values of HMM (15) and is consistent with HMM being monodisperse in solution.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Physical characteristics of purified
HMM. A, sedimentation coefficient for HMM (7.07 s) determined by sucrose gradient
ultracentrifugation. Standards were thyroglobulin (19.2 s),
catalase (11.2 s), aldolase (7.3 s), and albumin
(4.6 s). Inset, 12% SDS-PAGE of HMM produced by
-chymotryptic digestion of myosin. Labels indicate the positions of
HMM heavy chain (HC) and myosin light chains
(LC1, LC2, and
LC3). B, determination of Stokes radius
of HMM (130 Å) by gel filtration chromatography on a Superose 6 column. Standards were albumin (35.5 Å), catalase (52.2 Å), ferritin
(61 Å), thyroglobulin (85 Å), and laminin (186 Å).
|
|
Scattering Measurements--
Fig.
2A shows the neutron
scattering intensity profiles obtained for HMM in solutions containing
EDTA with and without added Ca2+ or Mg2+. The
merged data sets for each condition extend over the q range 0.004-0.24 Å
1. Guinier regions for each data set are
shown in Fig. 2B. Each shows evidence for two linear
regions, the larger slope associated with the Rg
value for the overall shape and the lesser slope associated with the
head group region. The rollover of the data at the lowest q
values is expected for asymmetric, rod-like particles of finite length.
Fig. 2C shows the pair-vector distribution functions,
P(r) versus r, obtained via
Fourier transform of the data using the GNOM software package. The
P(r) profile for each data set exhibited two
prominent maxima, one at r = ~37 Å and one at
r = ~110 Å. The peak at r = 37 Å is
a consistent feature of P(r) profiles derived
from solution scattering measurements of myosin S1 subfragments (29,
30) and, therefore, is likely to reflect interatomic vector lengths
within the S1 head. The longer vector lengths most likely reflect
contributions from both head-head and head-rod scattering vectors (see
below). The P(r) functions were similar
for all buffers, suggesting that the overall conformation of HMM was
not measurably different in buffers with or without added divalent
cations. The length at which the P(r) function
goes to zero gives the maximum chord length of the molecule, dmax. All three data sets were fit well at a
dmax of 390-400 Å and radius of gyration
(Rg) values of 110-112 Å using neutron scattering
data with a qmin of 0.004 Å
1. A
dmax value of ~400 Å is consistent with
electron microscope observations that indicate a sharp bend in the
myosin rod 43 nm from the head-tail junction (11). Flexibility of the
rod at this position and limitations from qmin
could contribute to an apparent foreshortening from the longest
estimated lengths of the S2 rod (72 nm) (31).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 2.
Small-angle scattering by HMM.
A, basic neutron scattering curves of HMM in experimental
buffers. Scattering data were collected at two separate detector
distances (13.1 and 1.7 m), background-subtracted, and merged in
the region of overlap. Data, reduced to
log[I(q)] versus q, are
shown for HMM in solutions containing 1 mM EDTA
(closed circles), 1 mM EDTA + 2 mM
MgCl2 (open circles), and 1 mM EDTA + 2 mM CaCl2 (squares). Data points
for the three solutions essentially superimpose; closed
circles and square curves were arbitrarily shifted
along the vertical axis for clarity. B, Guinier
regions for HMM in solutions containing 1 mM EDTA
(closed circles), 1 mM EDTA + 2 mM
MgCl2 (open circles), and 1 mM EDTA + 2 mM CaCl2 (squares). Open
circle and open square curves were arbitrarily shifted
along the vertical axis for clarity. C,
P(r) transformations of the data shown in
A obtained using GNOM software. D, concentration
dependence of Rg determined from x-ray scattering
measurements. Rg values were calculated from
scattering data using GNOM software.
|
|
X-ray scattering measurements were used to obtain plots of
Rg versus concentration, c, to
extrapolate values of Rg at infinite dilution (Fig.
2D). For these data scattering was corrected for effects of
slit smearing (see "Materials and Methods"), and
Rg values were again determined from
P(r) analysis. As expected for monodisperse
particles, the Rg versus c
relations showed either no concentration dependence or showed that
Rg decreased with increasing c, which is
characteristic of interparticle interference. The difference between
the Rg of HMM in solutions containing EDTA and in
solutions containing free added Mg2+ or Ca2+ is
not statistically significant, consistent with our conclusion from the
neutron scattering data that the addition of divalent cations does not
cause a large scale redistribution of molecular mass, i.e.
the disposition of the head groups with respect to each other and to
the rod are similar for each form.
I0 values obtained from
P(r) analysis of the x-ray scattering data were
used to calculate molecular weight values by comparison with lysozyme
as a standard (20). Molecular weight values calculated from the x-ray
scattering data underestimated published values for chymotryptic HMM by
as much as 25%. The underestimate was likely due to the large
dimensions of HMM combined with our limited ability to collect x-ray
scattering data below 0.01 Å
1, which are required to
adequately sample the long vector lengths within the particle. Indeed
the P(r) profiles calculated from the x-ray
scattering data go to zero at shorter vector lengths (~270 Å),
effectively truncating the vector lengths associated with the long tail
of the HMM molecule.
Model Structures--
To determine whether the existing model of
the head-tail junction of myosin (13) could adequately account for
experimental scattering by HMM, neutron scattering profiles were
compared with simulated spectra from a similar model. The original
model was constructed by aligning the crystal coordinates of two
scallop regulatory domains with model coordinates for a scallop
-helical coiled-coil rod. The remainder of the catalytic domain was
generated by the addition of chicken S1 coordinates (1). According to the resulting structure, the two S1 heads are predicted to lie in a
plane nearly anti-parallel to one another, with heads overlapping at
the distal ends of the light chain binding domains. The structure has
2-fold rotational symmetry about the axis of the coiled-coil tail.
Scattering intensity profiles were generated from the model using an
algorithm previously described (27). Intensity profiles were also
generated from models with coiled-coil tails of varying lengths (see
"Materials and Methods"). Fig.
3A shows one such model (tail
length = 380 Å) and a comparison of P(r)
functions (Fig. 3B) derived from the model to that obtained
from experimental scattering data (neutron scattering data from HMM in
solutions containing EDTA). Although the maxima at r = ~37 and 110 Å in the experimental P(r) were
reproduced reasonably well by the simulated data, the relative
intensities of the two peaks differed between the experimental and
model P(r) functions. The functions also differed
at a third maximum (r = ~200 Å), with the model
P(r) function giving a much more pronounced peak
compared with the experimental data function. The comparisons indicate
that significant differences exist between the published HMM model (13)
and HMM conformation in solution.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 3.
Comparison of a theoretical
P(r) function calculated from a model
HMM structure with that calculated from neutron scattering data.
A, wire-frame model of an HMM model similar to that
developed by Offer and Knight (13). B, comparison of
simulated P(r) functions (gray) from
the model shown in A with P(r) data of
HMM in buffers containing EDTA (black).
P(r) functions are shown on a relative scale such
that the area under each curve is proportional to the molecular weight
of the scattering particle.
|
|
Three independent best-fit models produced by allowing the two S1 heads
to freely rotate relative to the S2 tail are shown in Fig.
4. As can be seen from the wire frame
models, all three of the structures obtained using this approach
exhibited similar features, although two of the models differed from
the third with respect to chirality (handedness) of the heads. Because
small-angle scattering cannot distinguish between equivalent
conformations of different handedness, neither conformation can be
excluded. Compared with the starting model (Fig. 3A), the S1
heads in the optimized models are less planar and show marked asymmetry
with respect to the rod. Fig. 5 shows the
P(r) functions of the models compared with that
derived from experimental neutron scattering data. All three models
reproduce the initial peak at r = ~37 Å and
approximate the relative intensity of a second maximum. However, the
position of the second maximum shifts to shorter vector lengths (r = ~87 versus 110 Å). A third peak at
r = ~176 Å is also more prominent in the model
P(r) functions.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Wire-frame representations of three best-fit
HMM structural models. Top, longitudinal view.
Middle, view as in top panels but rotated
~90° counterclockwise. Bottom, view down rod axis. Model
1011 (red), F = 1.98; Model 1508 (green), F = 2.09; Model 1608 (blue), F = 2.16.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Comparison of theoretical
P(r) functions calculated from
best-fit HMM structural models using PR_PDB. Neutron scattering
data is shown in black, model 1011 is shown in
red, model 1508 is shown in green, and model 1608 is shown in blue. P(r) functions were
generated from the scattering data using GNOM software, and the area
under each curve was normalized to 1.
|
|
P(r) functions derived from the breakdown of one
of the models produced by XTAL_STR into its component parts are shown
in Fig. 6. According to this analysis,
the three broad maxima in the P(r) profile of the
complete model can be deconstructed into four peaks arising from model
subcomponents. The first and second of these, occurring at
r = 37 and 85 Å, are due primarily to scattering lengths within the individual heads and between the individual heads
and the rod, respectively. The third and fourth, at r = 115 and 176 Å, are due to vectors corresponding to distances between the two S1 heads. Potentially, the ratio of the first and second peak
intensities could be affected by segmental flexibility of the rod and
account for divergence of vector lengths among model and experimental
data in this region. Similarly, flexion of the individual heads at the
junction between the catalytic and light chain binding domains (29)
could modulate peak intensity at r = 176 Å. However,
these additional points of flexibility were not incorporated into the
current modeling because they would have introduced an unacceptable
number of degrees of freedom into the modeling.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
P(r) functions
derived from component parts of Model 1011. Solid line,
complete model; dashed line, up head and down head;
dotted line, down head and rod; dashed and dotted
line, up head and rod.
|
|
 |
DISCUSSION |
The results presented here are the first to use small-angle x-ray
and neutron scattering measurements to produce a model of the
three-dimensional structure of a double-headed myosin fragment in
solution. In contrast to previous studies of HMM by small-angle x-ray
scattering (32, 33), we utilized available S1 crystal structures to
construct HMM models that satisfy experimental scattering profiles of
HMM in solution. Although equivalent small-angle scattering intensity
profiles do not necessarily imply a unique structural solution, the
ability to obtain reproducible fits using constraints of known crystal
structures suggests that the models described here are reasonable
representations of the three-dimensional shape of two-headed myosin in
solution. The major new finding of this work is that the head-tail
junction of HMM is not symmetric.
Although it has been appreciated that the two heads of myosin may adopt
defined conformations with respect to one another, especially within
the thick filament (34-39), and that such relationships are likely to
have regulatory significance (40, 41), it has proven difficult to
define those interactions in isolated myosin. For instance, most
electron micrographs of myosin or HMM show that the heads adopt random
angles with respect to one another and to the rod (11, 42, 43). These
and other data suggest that both the S1-S2 junction and the junction
between the catalytic and regulatory domains of S1 are points of
flexibility in myosin (44). On the other hand, the notion that the two
heads of myosin adopt conformations independent of one another was
recently called into question by a model reconstruction of the
head-tail junction of myosin (13). The model was constructed by docking
a crystal structure of the scallop regulatory domain (2) to that of a scallop coiled-coil helix. Assumptions made in docking the structures were that the WP helix (i.e. the "hook" helix) of
the scallop regulatory domain was continuous with the
-helix of the
coiled-coil tail and that the entire repeating heptad sequence of the
tail forms a helical coiled-coil. According to the resulting model the
two S1 heads were predicted to emerge at angles tangential to the
myosin tail, lie anti-parallel to one another, and be related to each
other by a 2-fold axis of symmetry. Furthermore, based on steric
considerations and the results of molecular dynamics simulations, the
positions of the two heads relative to one another were predicted to be
relatively static (13).
Our current results demonstrate that simulated scattering from an HMM
model comparable with that of the head-tail junction of scallop HMM
(13) does not reproduce scattering by HMM in solution. In particular,
the third maximum P(r) function of the model was
not present in the P(r) function calculated from
the data. Calculation of the P(r) functions for
model subcomponents show that this third maximum is related to the
position of the heads relative to one another. The results indicate
that the average relationship of the two S1 heads differs from that
predicted by the model of the scallop head-tail junction (13).
Several explanations for the difference between experimental and
predicted observations are possible. First, the original model was
based on a chimeric HMM molecule composed of crystal structures from
both scallop and chicken S1 domains, whereas our experimental
scattering was obtained from purified rabbit fast skeletal HMM.
Although it may be reasonable to expect that scattering from the
chimeric HMM (probably closest to scallop HMM in the "on" state)
might resemble vertebrate striated HMM, this is not necessarily the
case. Second, conditions under which the experimental scattering data
were collected may also influence the conformation of the heads
relative to each other. The presence or absence of divalent cations did
not appear to affect scattering intensity profiles in the current
study. However, temperature (40), ionic strength (45), and the absence
of added nucleotides (29, 46) may still affect HMM conformation and
could account for differences between the experimental and model
scattering. Alternatively, differences between the experimental and
predicted HMM scattering profiles may be indicative of systematic
differences between model predictions of the head-tail junction and the
actual disposition of myosin heads in solution. In this case, the
XTAL_STR models obtained by allowing the S1 heads to rotate relative to
one another and the rod may be more representative of the average shape
of HMM in solution.
Characteristics of the optimized models that distinguish them from the
original representation of the myosin head-tail junction include a
marked departure from symmetry, a tendency for the light chain binding
domains to be related by angles of less than 180° (as opposed to
anti-parallel), and a tendency for one of the two S1 heads to be bent
back toward the S2 rod. Although these properties clearly differentiate
the optimized models from the starting model, they agree well with
electron micrograph representations of HMM (11, 42, 43). The lack of
symmetry between the heads is also consistent with findings from
2-dimensional cryoelectron projections of smooth muscle HMM in both the
thiophosphorylated ("on") and unphosphorylated ("off") states
(5, 47). Although it could be argued that asymmetry in the latter
models was artificially imposed by the packing of HMM molecules into a
two-dimensional lattice, our results indicate that S1 head asymmetry
persists even in solution.
The notion that the myosin heads exhibit structural and functional
asymmetry due to steric constraints within the thick filament or upon
actin binding has been suggested (37-39). For instance, although it is
well established that each S1 head of myosin, when isolated by
proteolytic digestion, is functionally identical to the other head with
respect to ATPase activity and nucleotide and actin binding affinities,
it seems likely that some nonequivalent behavior is induced by actin
binding. This is because in order for both heads to bind to actin in
the same filament either unwinding of a portion of the rod is necessary
or distortion of the second S1 head is required (7). Cooperative or
ordered binding may result (8) and could potentially explain the
greater force and displacement observed for HMM compared with S1 in
single molecule experiments (9).
Whereas asymmetry due to steric considerations may be imposed on the
myosin molecule, the present results suggest that differences in the
orientations of the two S1 heads are intrinsic to the structure of the
myosin dimer (48). At present, however, the molecular basis for
asymmetry between heads is speculative since the positions of the pivot
points linking the S1 heads to the tail were arbitrarily chosen for the
current modeling. Nonetheless, by combining molecular dynamics
simulations with the model of the scallop head-tail junction, Offer and
Knight (13) identified three points of flexibility within the molecule,
two within the long
-helix of the S1 regulatory domain and one in
the coiled-coil tail, which allow the two heads to move with respect to
the tail. Potentially, flexion at any of these points could contribute
to the overall disposition of myosin heads in solution. Alternatively,
flexibility at the WXW motif, which confers the sharp bend near the end
of S1 regulatory domain (1) or unwinding of the coiled-coil tail near
the invariant proline (12, 49), could also account for asymmetric head position.
In summary, we used small-angle scattering to investigate the
three-dimensional shape of HMM in solution. Comparison of the experimental scattering profiles to models of the head-tail junction of
HMM showed that the scattering profiles were most closely fit by models
in which the positions of the two S1 heads were nonequivalent and
asymmetric. It will be of interest to determine whether the disposition
of the S1 heads in HMM is affected by the presence of added nucleotides
or nucleotide analogues. If so, small-angle scattering techniques
combined with high resolution models of HMM will continue to prove
valuable for understanding interactions and contributions of both S1
heads during the catalytic cycle of myosin.