Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037
Brush border myosin-I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751-753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain-binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The ~31° swing corresponds to ~63 Å at the end of the rigid light chain-binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.
BRUSH border myosin-I (BBM-I)1 was the first vertebrate, unconventional myosin to be discovered
and is representative of one of the more abundant
classes of the myosin superfamily, the myosins-I (Pollard
et al., 1991 The most common isoform of BBM-I consists of a conserved myosin catalytic domain, a light chain-binding domain (LCBD) with three associated calmodulin (CaM)
light chains, and a COOH-terminal, lipid-binding domain.
The LCBD consists of three tandem repeats of a 23-residue
"IQ motif," so called because of their consensus sequence,
IQxxxRGxxxR (Cheney and Mooseker, 1992 Despite the growing importance of unconventional myosins, little structural information for them exists. Recently, we have begun to characterize the three-dimensional (3D) structure of BBM-I using EM. Cryo-EM of
actin filaments decorated with BBM-I in the absence or
presence of 10 mM MgADP revealed an ADP-dependent conformational change in BBM-I (Jontes et al., 1995 We have also extended the observation on an ADP-
induced conformational change in BBM-I; actoBBM-I, in
the presence of 1 mM MgADP, was found to swing through
an angle of ~31°, in agreement with the previous study
that had used 10 mM MgADP (Jontes et al., 1995 Protein Purification
BBM-I was prepared as described by Collins et al. (1990) Actin was isolated from rabbit skeletal muscle by the method of Spudich and Watt (1971) Specimen Preparation
BBM-I was dialyzed into 10 mM imidazole, pH 7.5, 50 mM NaCl, 1 mM
DTT, 1 mM EGTA, and 1 mM MgCl2. For the ADP experiment, BBM-I
in the above buffer was incubated with 1 mM ADP. Actin (diluted in the
above buffer) was applied to carbon-coated, copper EM grids (400 mesh)
at a concentration of 20 µg/ml (0.5 µM). After ~2 min, the grids were
rinsed with two drops of buffer and BBM-I was applied to the grids.
BBM-I concentrations were 0.5-1.0 mg/ml (3-6 µM) for the rigor experiments and 1.5-2.0 mg/ml (7.5-12 µM) BBM-I for the ADP experiments.
After ~2 min, the grids were blotted and plunged into ethane slush. Grids
were stored under liquid nitrogen.
EM
Grids were mounted in a Gatan (Pleasanton, CA) 626 cryo-stage and inserted into a Philips CM120 transmission electron microscope (Eindhoven,
Netherlands) operating at an accelerating voltage of 100 kV. Images were
collected at a nominal magnification of 35,000 and at defocus values ranging from Image Processing
Images were screened on an optical diffractometer for both image and filament quality. Selected images were required to be free of drift and astigmatism, and at appropriate defocus. Filaments showing good optical diffraction (symmetric about the meridian with intensity on the J2, J4, J The images were scanned on a flatbed scanning microdensitometer
(PDS 1010G; Perkin-Elmer Corp., Norwalk, CT) at spot and step sizes of
20 µm, corresponding to 5.71 Å at the specimen. The filaments were subsequently processed using the PHOELIX helical image processing package (Whittaker et al., 1995a Statistical Analysis
The individual data sets were moved to a common phase origin and maps
were calculated. A mean density and variance were calculated for each
voxel in the 3D maps of both the rigor and ADP data sets. A Student's t
test was then used to compare these two structures (Milligan and Flicker,
1987 Images and Image Analysis
Fig. 1 shows two cryoelectron micrographs of actin filaments
fully decorated with purified BBM-I in the absence (Fig. 1
a) or presence (Fig. 1 b) of 1 mM ADP. These filaments do
not show the characteristic arrowhead appearance of actin
filaments decorated with skeletal muscle myosin subfragment-1 (S1) (Milligan and Flicker, 1987
Table I.
Image Processing of Actin Filaments Decorated
with BBM-I
The actoBBM-I Rigor Complex
The averaged layer line data for the rigor filaments were
truncated to 30 Å and used in a Fourier-Bessel synthesis to
produce the 3D map presented in Fig. 3. The BBM-I molecule extends out from the actin filament and displays density
that can be interpreted in terms of three domains: the myosin catalytic (or motor) domain, the LCBD, and the lipid-binding domain (Fig. 3). The catalytic domain is a large,
globular density that binds tangentially to the actin filament,
whereas the LCBD is a long density extending out orthogonally from the filament axis (Whittaker and Milligan, 1997
Pseudo-atomic Model
We used the crystal structure of the skeletal muscle myosin
catalytic domain and the associated ELC to build an approximate atomic model of BBM-I (Rayment et al., 1993a Similarly, three copies of the ELC crystal structure were
fit into the LCBD (Fig. 4). The ELC was used, since Houdusse et al. (1996) Once the fitting was performed, it was then possible to
compare the orientation of the BBM-I LCBD to that of
skeletal muscle myosin (Fig. 5). The S1 HC crystal structure
was placed in the same orientation as the BBM-I model
and the HC
The Ternary Complex of Actin-BBM-I-MgADP
Fig. 6 presents the 3D map calculated from the layer line
data shown in Fig. 2 b. Consistent with our previous study
(Jontes et al., 1995 In stark contrast to the catalytic domain, the LCBD of
BBM-I has adopted a completely different conformation.
The entire LCBD appears to have rotated as a rigid body
(Fig. 8). Jontes et al. (1995)
Rigor Complex
The actoBBM-I rigor complex we have visualized by cryo-EM is similar to previously characterized myosins (Milligan and Flicker, 1987 As has been discussed previously (Jontes and Milligan,
1997 The Lipid-binding Domain
It is clear from comparing BBM-I in the presence and absence of ADP that the angle of the LCBD relative to the
lipid-binding domain (or the plasma membrane) has to
change during a BBM-I powerstroke. This change requires
that the junction between the light chain 3 (LC3) and the
lipid-binding domain act like a hinge. It has been argued
previously that this might be the case in order for BBM-I
to attach to actin filaments that approach the membrane from different angles (Jontes and Milligan, 1997 Effect of MgADP
The reorientation of the LCBD in the presence of 1 mM
MgADP reveals a dramatic effect of MgADP on the conformational equilibrium of BBM-I (Fig. 8; and Jontes et
al., 1995 Whereas BBM-I clearly undergoes a large conformational change in response to ADP binding, it is not clear
which biochemical state in the ATPase cycle has been
trapped. Incorporation of our structural results into the
existing framework of actomyosin mechanochemistry requires identifying to which step in the cycle our ADP state corresponds. Analysis of the skeletal muscle myosin and
actomyosin ATPases has demonstrated that ADP binding
and release occurs in at least two steps: a slow isomerization and a rapid equilibrium association/dissociation (Bagshaw and Trentham, 1972; Sleep and Hutton, 1980 For skeletal muscle myosin, addition of ADP to AM is insufficient to drive the ternary complex through step 2;
since K2 Involvement in Force Production
Given the above interpretation, the question can be asked:
coming forward through the ATPase cycle, will reversal of
the ADP-dependent conformational change (Fig. 8 b) result in force production and contribute to a myosin working stroke? On the basis of structural results alone, it is not
possible to answer this question, although the movement
closely matches predictions based on a variety of structural data (Huxley and Kress, 1985 The experiments most directly relevant to the mechanism of force generation have been mechanical experiments
performed on muscle fibers. If a muscle fiber is stimulated
to contract isometrically in the presence of added Pi, the
steady-state force is reduced (Cooke and Pate, 1985 The results of these mechanical studies make it appear
very unlikely that the 63-Å movement we observe represents a force-generating transition analogous to that observed in skeletal muscle fibers, although it is not clear
what event(s) the mechanical transients are actually measuring. This leads to a second possibility, first suggested by
Huxley and Simmons (1971) with the assignment of our ADP-state to the AM
Variation within the Myosin Superfamily
Our structural results also create an apparent paradox:
while we have trapped an apparent intermediate in the
force-generating cycle, our ability to trap this state brings
into doubt its ability to generate force. In other words, the
accessibility of this state suggests that the free energy barrier between this state and rigor are relatively small. In
turn, this small free energy difference indicates that relatively little work can be obtained at this step. Conversely,
in the case of skeletal muscle myosin the large free energy
difference between AM The large free energy drop between AM.ADP states of
skeletal muscle myosin could represent an adaptation of
this myosin, reflecting the functional requirements of fast
skeletal muscle. Destabilization of the AM If it is the case that we have visualized the AM; Hammer, 1994
; Mooseker and Cheney, 1995
). Originally identified in the microvillus of intestinal epithelial cells as lateral bridges linking the core actin bundle to
the plasma membrane (Matsudaira and Burgess, 1979
; Howe
and Mooseker, 1983
), BBM-I has been shown to be a functional myosin motor protein, having actin-activated ATPase and in vitro motility activities (Collins and Borysenko,
1984
; Conzelman and Mooseker, 1987
; Collins et al., 1990
;
Wolenski et al., 1993
).
; Titus, 1993
; Wolenski, 1995
). A minor isoform contains a 29-residue
splice insert, resulting in a fourth IQ motif (Halsall and
Hammer, 1990
). The lipid-binding domain consists of a region rich in basic amino acids (Garcia et al., 1989
), and has
been shown to mediate binding to anionic phospholipid
vesicles (Hayden et al., 1990
).
). Additionally, Whittaker and Milligan (1997)
have used actoBBM-I to investigate conformational changes in the LCBD
in response to calcium. Unfortunately, in both of these studies only ~75% of the protein was visualized; no density was
observed that could be attributed to either the third calmodulin light chain or the COOH-terminal, lipid-binding domain. In a separate study, tilt-series reconstruction of
two-dimensional crystals of BBM-I was used to calculate a
3D map (Jontes and Milligan, 1997
). Whereas density
could be assigned to each of the major structural domains
of BBM-I, the exact boundaries of the catalytic domain
and the position of the actin-binding site could not be identified with certainty. Here, we use cryo-EM of actoBBM-I to extend the previous observations of Jontes et al.
(1995)
, visualizing the entire BBM-I molecule. We have
also generated a pseudo-atomic model of BBM-I by fitting
the crystal structures of the skeletal muscle myosin catalytic domain and the skeletal muscle myosin essential light
chain (ELC) into our EM envelope (Rayment et al., 1993a
).
Thus, we have provided the most detailed structural information available for any unconventional myosin.
). Since
we now see the entire LCBD, we measure the magnitude of the movement to be ~63Å. In addition to the axial
translation, the LCBD also appears to rotate about its long
axis by 20 to 30° during the transition from the rigor state
to the ADP state. We also discuss the potential relevance
of the ADP-dependent movement to actomyosin (AM)
force production, as well as its relevance to possible variation in mechanochemistry across the myosin superfamily.
Materials and Methods
with a few modifications. Briefly, the small intestines were excised from six female White
Leghorn chickens, and were split lengthwise, cut into 5-6-in segments, and
then washed in 10 mM imidazole, pH 7.3, 140 mM NaCl. The brush borders were released by stirring in 10 mM sodium phosphate, pH 7.3, 10 mM
EDTA, 140 mM NaCl, 100 mM sucrose, 2 mM PMSF for ~60 min at room
temperature. The intestines were then rubbed vigorously to extract as
much of the epithelial layer as possible. The initial extract was centrifuged
in a JA-4.3 rotor (Beckman Instuments, Inc., Palo Alto, CA) for 10 min at 4,000 rpm. After homogenization in 10 mM imidazole, pH 7.3, 4 mM
EDTA, 5 mM EGTA, 0.2 mM Pefabloc-SC (Boehringer Mannheim, Mannheim, Germany), 1 mM PMSF, 5 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml aprotinin using a polytron blade homogenizer, the isolated brush
borders were spun down (JA-4.3 rotor, 4,000 rpm for 15 min at 4°C), and
rinsed twice in 10 mM imidazole, pH 7.3, 75 mM NaCl, 5 mM EDTA, 2 mM EGTA, and 0.2 mM Pefabloc-SC, 1 mM PMSF, 5 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml aprotinin by homogenization and pelleting
(JA-4.3 rotor, 4,000 rpm for 15 min at 4°C). The brush border pellet was
resuspended in 20 mM imidazole, pH 6.9, 2 mM EGTA, 20 mM MgCl2,
400 mM NaCl, 5 mM ATP, 1 mM DTT with 0.2 mM Pefabloc-SC, 1 mM
PMSF, 5 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. The pellet was resuspended by homogenization in a loose-fitting dounce, and another 5 mM ATP was added. The homogenate was spun in a Ti70 rotor
(Beckman Instuments, Inc.) at 28,000 rpm for 30 min, and loaded onto
two identical Sepharose CL-4B gel filtration columns. Fractions containing BBM-I were pooled and dialyzed overnight into 10 mM imidazole, pH
7.5, 50 mM NaCl, 2 mM EGTA, 0.1 mM MgCl2, 10% sucrose with 0.2 mM
Pefabloc-SC, 1 mM PMSF, 5 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml
aprotinin. The dialysate was loaded onto a CM-Sepharose cation exchange column and step eluted with starting buffer containing 500 mM
NaCl. Fractions containing BBM-I were pooled, diluted ~fourfold, and
then loaded onto a 1 ml MonoQ anion exchange column. BBM-I was eluted
with a 50-500 mM NaCl gradient. Fractions containing BBM-I were then pooled and diluted fourfold into the same 50 mM NaCl buffer, loaded onto a MonoS cation exchange column and eluted with a 50-500 mM NaCl
gradient.
.
1.4 to
2.1 µm.
3,
J
1, J1) were selected for densitometry and computer processing.
; Carragher et al., 1996
), using the MRC (Medical Research Council, Cambridge, England) suite of programs (DeRosier
and Moore, 1970
). Briefly, each filament was straightened, the positions of
the layer lines were determined, and an integral number of repeats was
excised and floated into an array size suitable for Fourier transformation.
The positions of zeroes in the contrast transfer function (CTF) were determined for each image. Each image was corrected for the effects of the CTF assuming 10% amplitude contrast. Layer line data were taken from
the transform after refinement of the filament axis position. The data from
the individual filaments were then fit together and averaged using an individual data set as an initial template. The fitting and averaging were integrated using the previous average as a template until the averaged layer
line data ceased to change between cycles (usually three to four cycles).
Fitting was performed using the peaks of the strong Bessel orders (J = 2, 4,
5,
3,
1, 1). The final averaged data set was then used to "sniff" the transforms of the individual data sets (Morgan et al., 1995
). The sniffing
procedure discards the assumption of perfect helical symmetry and uses
the averaged data to refine the layer line position. This procedure results
in an improved signal-to-noise ratio on weak layer lines. The sniffed data
sets were then fit and averaged in two cycles of averaging. The final data
set was truncated to 30 Å and used to calculate a 3D map in a Fourier-Bessel synthesis. Solid, 3D surfaces were rendered using the program
SYNU (Hessler et al., 1992
). Fitting of atomic models to EM maps was
performed manually using the program O (Jones et al., 1991
).
). Differences of P < 0.0001 were presumed to be statistically significant.
Results
). This difference is
likely due to the fact that BBM-I extends out nearly orthogonally from the actin filament (see below), as well as
to the much higher background of BBM-I preparations
relative to those of myosin S1. Fig. 1, c and d, shows the
computed transforms of the images in a and b, respectively. Although the images have rather low contrast, both
the rigor and ADP filaments give reasonably sharp, although weak, diffraction patterns. 42 images of rigor filaments and 23 images of the actoBBM-I filaments, decorated in the presence of 1 mM ADP, were analyzed to
produce the layer lines shown in Fig. 2. The layer lines
shown have been truncated to 30 Å and represent all of
the data used to synthesize the 3D map (below). The layer
line data for the two averages are very similar with only some
minor differences noticeable on the Bessel orders n = 4,
3, and 1, for example. A summary of the parameters obtained from the fitting and averaging is presented in Table I.
Fig. 1.
Images of actin filaments decorated with BBM-I. (a)
A cryoelectron micrograph of actoBBM-I in the absence of nucleotide (rigor), which has been digitized and computationally
straightened. There is very low contrast, due to an excess amount
of protein in the background. (b) A cryoelectron micrograph of
an actin filament decorated with BBM-I.ADP. As with the rigor
images, actoBBM-I.ADP exhibits very low image contrast, due
primarily to a high background. (c and d) Computed Fourier transforms of the straightened filaments shown in a and b, respectively.
[View Larger Version of this Image (58K GIF file)]
Fig. 2.
The final layer line data for actin filaments decorated
with BBM-I. The layer line data in a and b were used in the Fourier-Bessel synthesis of the rigor and MgADP maps shown in
Figs. 3 and 6, respectively. The data were truncated to a uniform
resolution of 30 Å. The solid lines represent the amplitudes and
the dotted lines represent the phases for each layer line. The ordered pairs of numbers are the Bessel order (n) and the layer line
number (l) for a 54:25 helical selection rule.
[View Larger Version of this Image (35K GIF file)]
Fig. 3.
3D map of actoBBM-I in the absence of nucleotide, calculated from the layer line data shown in Fig. 2. Densities can be
identified in the 3D map that are attributable to the catalytic domain, each of three calmodulin light chains, and a lipid binding
domain, as indicated. C, catalytic; 1, 2, and 3, indicate the three
calmodulin light chains; LB, lipid-binding domain.
[View Larger Version of this Image (103K GIF file)]
Fig. 6.
3D map of actin filaments decorated with BBM-I.ADP.
The map was calculated using the layer line data shown in Fig. 2 b.
Like the rigor map, mass can be seen to extend to high radius. The gross features of both the rigor and ADP maps are essentially the
same: a globular catalytic domain and a long, irregular light chain-
binding domain. Although the lipid-binding domain is not visible
in the ADP map, mass protrudes from the end of the light chain-
binding domain, at the position where the lipid-binding domain is
found in the rigor map. The large conformational change is quite
striking (compare to Fig. 3), giving the impression that the filament has reversed polarity. C, catalytic domain; 1, 2, and 3 indicate the three light chains; LB, the lipid-binding domain.
[View Larger Version of this Image (97K GIF file)]
).
The LCBD consists of three densities, which presumably correspond to the three bound CaM light chains (Figs. 3 and
4). The putative, lipid-binding domain can be assigned,
since it is known to be located at the COOH terminus, distal to the third CaM light chain. Additionally, its shape
and position closely match that of the lipid-binding domain assigned in the tilt-series reconstruction of negatively
stained crystals of BBM-I (Jontes and Milligan, 1997
). Thus,
consistent with the results of our earlier studies, the BBM-I
molecule is an elongated and irregularly shaped molecule.
The length of BBM-I as measured in the helical reconstruction is ~230-Å long, which closely matches the length found in the map calculated by tilt-series reconstruction
(Jontes and Milligan, 1997
). The details of the map strongly
suggest that the entire BBM-I molecule has been visualized.
Fig. 4.
3D model of BBM-I.
This model was built by fitting the x-ray structures of
the myosin catalytic domain
and the ELC of skeletal muscle myosin into the EM density map (magenta wire cage)
for BBM-I. At the COOH-terminal end of the molecule,
an extra density is found
which can be attributed to
the basic, lipid-binding domain. In addition to the catalytic domain, the three light
chains (LC1, LC2, and LC3)
and the lipid-binding domain (LB) are indicated. The
C backbones of the catalytic domain and the three
light chains are displayed in
alternating yellow and white
for clarity, and the HC helix
is shown in green.
[View Larger Version of this Image (61K GIF file)]
).
As has been done previously (Rayment et al., 1993b
; Jontes et al., 1995
; Whittaker and Milligan, 1997
), the backbone of the myosin catalytic domain was fit into the EM
density. Since the myosins-I lack the NH2-terminal
barrel present in myosin-II (Pollard et al., 1991
; Mooseker and
Cheney, 1995
), these residues were removed from the crystal structure before fitting.
have suggested that the structure of
apocalmodulin bound to an IQ motif should assume a conformation similar to that found in the light chains of muscle myosins. Because the shapes of the light chains and the
resolution of the EM map do not permit as unambiguous a
fit as was obtained for the catalytic domain, a number of
other constraints were used. First, it was assumed that the
axis of the heavy chain (HC) helix was oriented roughly parallel to the long axis of the LCBD, and that the COOH terminus of each HC helix should point away from the catalytic domain. Additionally, the 23-residue IQ motif suggests
that there should be a spacing of ~35-40 Å and a rotation
of ~140° between successive CaMs, assuming a perfect
-helix (3.6 residues per turn, and 1.5 Å/residue). The final
step was to manually fit the individual calmodulins into the
EM density to produce a "best-fit," as determined by eye.
Using these criteria, the fit shown in Fig. 4 was obtained. The first and second light chains fit the EM density reasonably well while obeying each of the fitting criteria. However,
the third light chain fit the EM density much better with a
rotation of only ~100°, rather than the predicted ~140°.
This may indicate that the HC helix is kinked or disordered at the junction between the second and third light
chains.
-helix was compared to that of BBM-I (Fig. 5
a). From these fits, it is apparent that the
-helix of BBM-I
exits the catalytic domain from a slightly different position
than in S1 and has a significantly different orientation. The
end of the HC helix of BBM-I is displaced upward by several angstroms relative to that of S1 (Fig. 5 b). Next we compared the orientation and path taken by the BBM-I
and S1 HC helices. The BBM-I HC helix projects out nearly
orthogonally from the filament axis, whereas the S1 helix
angles downward, defining the "barbed" end. Additionally, the BBM-I helix appears to remain quite straight
throughout the length of the LCBD; the S1 helix is much
more curved. Although this difference could partially be
due to the fitting procedure itself, this is not very likely, since maintaining a straight, colinear helix was not a constraint of the light chain fitting. Thus, this comparison appears
to highlight real differences in the two LCBDs, reflecting
both the differences in the HC sequences (including IQ
motif spacing) and the differences in light chain composition (ELC and regulatory light chains vs. CaM).
Fig. 5.
Comparison of the BBM-I
and skeletal muscle S1 light chain-
binding domains. (a) Fit of the myosin
catalytic domain (yellow), the BBM-I
LCBD helix (green), and the skeletal
muscle S1 light chain-binding domain
helix (white). There is clearly a difference in the position of the LCBD between the two myosins, indicating a
different exit point of the long -helix
from the catalytic domain. (b) A stereo
pair of the backbones shown in a, rotated ~90° about the filament axis. The
EM density has been omitted for clarity. The S1 helix also appears displaced
laterally relative to the BBM-I helix.
(c) A view of the carbon backbones,
looking down the filament axis, rotated
90° about the horizontal. Again, the
EM density was omitted for clarity.
[View Larger Version of this Image (52K GIF file)]
), the structure of the actoBBM-I complex in the presence of MgADP has a similar overall shape
to that of the rigor structure. As in the rigor structure, densities are found that can be attributed to the catalytic domain and the LCBD with its three associated light chains.
The lipid-binding domain can not be seen, although, at
lower contours mass begins to protrude from the end of
the third calmodulin (Fig. 6). The catalytic domain appears to be attached to actin in a manner identical to that
found in the absence of ADP. This conclusion is supported
by two lines of evidence. First, the same orientation of the
myosin catalytic domain fits both density maps equally
well. The x-ray fits of the catalytic domain shown in Figs. 4 and 7 are identical; the position of the catalytic domain has
not been altered between the separate EM maps. More
quantitatively, a statistical difference map calculated for
the two data sets does not reveal any significant differences in the catalytic domain (P < 0.0001), consistent with
the previous study of Jontes et al. (1995)
.
measured a change in orientation of ~32°, producing an angular movement of ~50 Å at
the end of the second light chain in response to 10 mM
MgADP. Here we have used the x-ray fits to the improved
maps to make more accurate estimates of the movement. The entire LCBD defined in Fig. 4 was rotated as a single
unit to provide the fit shown in Fig. 7. In addition to the
large axial swing, the fit was improved slightly by rotating
the LCBD about its long axis by 20-30° (Fig. 8 a). This additional rotation was also observed in our earlier work
(Jontes et al., 1995
), but the lack of distinguishing features
in the LCBD made it difficult to obtain an accurate estimate of its magnitude. The results of the fitting procedure
are consistent with the proposal that the LCBD rotates as
a rigid body, while the catalytic domain remains attached
to the actin in a fixed orientation. These results are summarized in Fig. 8 b.
Fig. 8.
Comparison of the ADP and rigor 3D maps. (a) Direct
comparison of the rigor and ADP maps clearly reveals the large
swing of the BBM-I LCBD. In addition to the swing, there also
appears to be a 20-30° rotation of the LCBD about its long axis.
The dotted and solid red bars represent the orientation of the
ADP (left) and rigor (right) LCBDs, respectively. (b) A superposition of the two x-ray fits for direct comparison of the two conformations. The common catalytic domain is in dark blue, the
ADP LCBD is in cyan, and the rigor LCBD is in magenta. Shown
in black are five monomers of the actin filament. This view highlights the magnitude of the BBM-I tail swing, as well as the fact
that the tail moves as a rigid unit.
[View Larger Version of this Image (57K GIF file)]
Fig. 7.
3D model of BBM-I
in the presence of 1 mM
MgADP. The EM density is
shown in magenta, the myosin catalytic domain is in yellow, the CaM light chains are
in cyan, and the HC helix is
shown in green. The results
of the rigor fitting (Fig. 4)
were rotated as a rigid unit to
obtain the fit to the ADP
map. The light chain-binding domain was rotated by ~31°
with respect to the rigor orientation, in addition to a rotation of 20 to 30° about its
long axis.
[View Larger Version of this Image (55K GIF file)]
Discussion
; Whittaker et al., 1995b), although it
also displays a number of substantial differences. Notably,
the geometry of actin binding by the catalytic domain is quite
similar to that of S1 (Whittaker and Milligan, 1997
), as might
be expected of such closely related proteins. However, the
molecular envelope of the BBM-I molecule differs significantly from that of conventional myosins-II (Milligan and
Flicker, 1987
; Whittaker et al., 1995b
). The catalytic domain of BBM-I lacks an NH2-terminal extension present
on myosin-II, which the chicken skeletal muscle (S1) crystal structure revealed to be a
barrel domain (Pollard et al.,
1991
; Rayment et al., 1993a
, b
; Whittaker and Milligan, 1997
).
More significantly, the LCBD extends out from the catalytic domain in an orientation nearly orthogonal to the filament axis, whereas the S1 LCBD extends out at a more
acute angle orientation (Milligan and Flicker, 1987
; Rayment et al., 1993b
; Whittaker and Milligan, 1997
), which is responsible for the traditional assignment of a 45° angle.
This difference can be attributed to the difference in position and orientation of the long, light chain-binding helix
(Fig. 5). A number of factors may contribute to the variation in LCBD position. First, the interactions of the HC
"converter" region with the proximal region of the LCBD
may differ depending on the type of light chain bound to
the first IQ motif, i.e., CaM vs. ELC (Houdusse et al., 1996
).
Additionally, the absence of an NH2-terminal extension in
BBM-I could also play a role in altering the position of the LCBD. Alternatively, the observed differences may be a
"delocalized" property reflecting overall differences in myosin sequence and structure.
), the BBM-I LCBD consists of three main densities
spaced ~35-40 Å apart, consistent with the spacing expected from the 23-residue IQ motifs. We fit the crystal
structure backbone of the skeletal muscle ELC into the
EM density for each of the three CaM light chains. Although a crystal structure exists for CaM bound to a target
peptide (Meador et al., 1992
), Houdusse et al. (1996)
have suggested that the conformation of apocalmodulin bound
to an IQ motif will more closely resemble that of a bound
ELC. Initial modeling of the CaM structure of Meador et
al. (1992)
produced a reasonable fit to the EM density
(data not shown), but we found that the ELC backbone fit
our map somewhat better. As can be seen, the fitting of
the light chains into the LCBD fills the EM envelope relatively well (Fig. 4). At the resolution of our 3D map, we are unable to make any meaningful statements about the
conformational states of the CaM light chains. However,
neglecting the details of CaM conformation, we feel the
fits are approximately correct and are sufficient to provide
a qualitative model of BBM-I structure.
). This
also suggests that the extent of the translational movement
should be measured from the end of the third light chain
(as was done here), not from the furthest density in the
map, as any structure beyond the hinge would not contribute significantly to the rigid "lever arm". Given the apparent flexibility in the junction between LC3 and the lipid-binding domain (Hayden et al., 1990
; Jontes and Milligan,
1997
), this junction could be a possible location for BBM-I
regulation. Swanljung-Collins and Collins (1992)
have provided some evidence for a regulatory mechanism involving
this part of the molecule; the BBM-I lipid-binding domain
is phosphorylated by protein kinase C and this phosphorylation is potentiated by binding to phospholipids. Additionally, recent work has shown that BBM-I displays reduced motility and actin binding while bound to lipids
(Zot, 1995
). It could be the case that phosphorylation of
the BBM-I tail affects the flexibility of the LC3-lipid-binding domain junction, possibly inducing a more upright posture of BBM-I on the membrane. In this regard, BBM-I
may act like skeletal muscle myosin where crossbridges lie
close to the thick filament during relaxation, and extend
away from the thick filaments during activation (Huxley,
1969
).
). The magnitude of the change in angle is ~31°,
resulting in an axial translation of ~63 Å. The LCBD appears to move as a rigid body, with the attachment to actin
remaining fixed. In addition to the axial translation, there
appears to be a ~20-30° rotation of the LCBD about its
long axis (Fig. 8 a). As in the earlier work of Jontes et al.
(1995)
, no significant differences were found in the catalytic domain between the rigor and ADP maps.
; Trybus
and Taylor, 1982
; Rosenfeld and Taylor, 1984
; Taylor, 1991
).
Ligand binding generally occurs in two steps: rapid formation of an initial complex followed by a rate-limiting isomerization (Fersht, 1985
; Gutfreund, 1995
). The ADP-bound state we have trapped could either be the initial
BBM-I-ADP, rapid equilibrium complex, or it could be
the state formed after a subsequent isomerization. The
first possibility suggests that the BBM-I tail would wag
with each rapid equilibrium ADP binding/release cycle. The
second possibility supposes that the observed isomerization occurs between ADP-bound states, a step distinct from
the binding reaction itself. Although our data cannot distinguish between these two possibilities, we favor the latter proposal for two reasons. First, it seems more plausible
that such a large structural rearrangement (Fig. 8 b) would
be associated with a slower isomerization step rather than with a rapid binding reaction. Second, our structural results can be more easily incorporated into the kinetic pathway with this interpretation. In the terminology of Sleep
and Hutton (1980)
, we have equated our ADP state with
the AM
.ADP state in the following scheme:
0.02 (Sleep and Hutton, 1980
), it would be impossible to trap the AM
.ADP state. While this appears to
argue against our interpretation, we emphasize that the
large free energy barrier to step 2 has not been shown to
exist for BBM-I. Skeletal muscle myosin gets trapped at
AM.ADP, but BBM-I and smooth muscle myosin may
have access to the AM
.ADP state. Consistent with this
scheme, no conformational change is observed in skeletal
S1 in response to ADP (Gollub et al., 1996
; Diaz-Avalos,
R., and R.A. Milligan, unpublished observations). If the
assignment of our structural state to AM
.ADP is correct, it would indicate a substantial difference in the kinetics
and energetics between BBM-I (and smooth muscle myosin)
and skeletal muscle myosin. Alternatively, skeletal muscle
myosin, smooth muscle myosin, and BBM-I might all be in
the AM.ADP state, which raises the difficult question of
why addition of ADP elicits a conformational change in
some myosins and not others. Detailed investigation of
BBM-I and smooth muscle myosin kinetics will be required to settle this issue.
; Cooke, 1986
; Vibert
and Cohen, 1988
).
). Similarly, initiation of active contraction in the presence of excess Pi accelerates the rise in tension, while decreasing the
final isometric tension (Hibberd et al., 1985
). These results
suggest that phosphate is able to bind to an AM
.ADP
state (believed to be the major force-producing state) and
to reverse the force-generating transition. This conclusion is supported by flash-photolysis studies. Photorelease of
caged Pi in isometrically contracting fibers causes an exponential decrease in tension (Dantzig et al., 1992
, Millar and
Homsher, 1992
; Homsher et al., 1997
), with the rate of decline showing a hyperbolic dependence on Pi concentration. These results have been interpreted to indicate that
binding of phosphate to a crossbridge induces a reversal of
the powerstroke. The mechanical evidence, as interpreted,
is most consistent with the proposal that the force-producing step occurs before Pi release and therefore, before the
state we have observed by EM.
, that the myosin working stroke
should occur in two or more steps. This proposal was originally based on the low values of work per crossbridge
which were obtained if the working stroke was assumed to
occur in a single step. Our cryo-EM results are consistent
with this proposal. Given the results from mechanical experiments and our EM study, it can be suggested that force
is produced in the following transitions:
.ADP
state of Sleep and Hutton (1980)
. This scheme would be
consistent with our structural results and with the mechanical experiments, while satisfying the prediction of Huxley
and Simmons (1971)
that the working stroke occurs in two
or more discrete steps. Ma and Taylor (1994)
, based on
myofibril kinetics, have also suggested that there may be
two force-producing transitions. A structural interpretation of this scheme is presented in Fig. 9. It is important to note that, in this scheme, neither structural transition occurs synchronously with a ligand release step; in both cases, a large
conformational change is stabilized by a subsequent biochemical step. Finally, it must be mentioned that the ADP
state we observe may be an additional step, having no analogue in the ATPase cycle of skeletal muscle myosin. However, we believe that the working hypothesis presented above
is the simpler interpretation.
Fig. 9.
Structural interpretation of the events occurring
during force production. This
is a graphic representation of
our interpretation of the force-
generating cycle, as presented
in the discussion. The transition from AMADP.Pi to
AM
.ADP.Pi is based on mechanical studies performed on
skeletal muscle fibers. The
representation of this as an
additional rotation of the
LCBD is purely hypothetical (emphasized by ?), as no
structural rearrangements
have yet been demonstrated.
In this scheme, each force-producing step can be considered to be a two-step
process: a force-generating
isomerization followed by a
ligand release step. This diagram also emphasizes that
force is not directly coupled
to products release, but occurs in separate steps. The
steps in the cycle leading from
rigor to the weakly bound
states have been omitted, as
indicated by the dotted arrow.
[View Larger Version of this Image (59K GIF file)]
.ADP and AM.ADP suggests that this step could produce work, yet this same energy difference prevents it from being visualized (Gollub et al., 1996
;
Diaz-Avalos, R., and R.A. Milligan, unpublished observations). A small free energy change for a given transition
would appear to limit the amount of work that can be extracted from that step. However, it has been pointed out
previously that free energy transduction is a property of
the cycle and cannot be attributed to individual steps (Hill
and Eisenberg, 1981
). In the case of BBM-I, the transition between AM
.ADP and AM.ADP could be pulled forward,
since any work performed during this transition would be
trapped by ADP release and the subsequent dissociation
of actomyosin by ATP (Gollub et al., 1996
). A detailed analysis of BBM-I kinetics and mechanics will be required to
provide further evidence for this suggestion.
. ADP state
would accelerate the flux through this part of the ATPase
cycle, reducing the amount of time spent attached to actin.
The increased rate would reduce the amount of drag exerted by attached, negatively strained cross-bridges and increase the maximal shortening velocity (Huxley, 1957
; Siemankowksi et al., 1985). Conversely, a smaller free energy
drop will make the step more reversible, reduce the Vmax,
and increase the time spent generating force. The latter case
could correspond to the situation of smooth muscle. Since
much of the free energy of ATP hydrolysis is derived from
the steady-state concentrations of ATP, ADP, and Pi maintained in muscle fibers (White and Taylor, 1976
), the accumulation of ADP would reduce the forward driving force
and slow the cycle. Similarly, most of the mechanical effects of ADP and Pi on contracting muscle fibers could be
explained simply in terms of limiting the flux into (Pi) and
out of (ADP) the force-producing states. These considerations may relate to the "latch" state of smooth muscle myosin (Hai and Murphy, 1989
), in which smooth muscle
sustains an elevated force, while ATPase activity decreases. A number of studies have suggested that this phenomenon may be a product of the higher affinity of
smooth muscle myosin for ADP, relative to skeletal muscle myosin (Drew et al., 1992
; Nishiye et al., 1993
). From
this point of view, the lack of a latch state in skeletal muscle would be a novel aspect of its kinetic cycle, allowing it
to achieve higher shortening velocities.
.ADP
state, our results indicate that the energy difference between ADP states is not as great for BBM-I as it is for
skeletal S1. This possibility suggests that although myosins
presumably operate by a fundamentally similar mechanism, there are substantial variations in the kinetics and
energetics of different myosins. Such variation complements the structural differences between myosins, as emphasized by Jontes et al. (1995)
. The magnitude of the angular swing is ~50% greater for BBM-I than for smooth
S1, ~31° vs. ~23° (Whittaker et al., 1995b
). This corresponds to an 80% increase in the step size, ~63 Å vs. ~35
Å. Additionally, as mentioned above, a number of the details also differ between these two myosins; smooth muscle S1 appears to lack the rotational component of the movement found in BBM-I, and BBM-I appears to lack the statistical differences found in the smooth S1 catalytic domain. Although the nature of the movement appears to be
roughly conserved, the response of these myosins to ADP
differs both qualitatively and quantitatively. It becomes
clear that results obtained with one myosin are not necessarily relevant to other myosin family members. This diversity in kinetic, structural, and mechanical properties
could allow for a more complete understanding of myosin
function, since detailed analysis of only a single myosin
type may miss salient features of the actomyosin mechanism that may not be apparent or experimentally accessible. A stronger thesis suggests that a full understanding of
the myosin mechanism can only be obtained through this
type of comparative analysis. It is expected that the distribution of myosin properties will reflect a corresponding
range of functional requirements, as myosins of different
shapes and sizes fulfill a variety of roles in the daily economy of cells and organisms.
Received for publication 18 June 1997 and in revised form 21 August 1997.
Address all correspondence to R.A. Milligan, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Tel.: (619) 784-9827. Fax: (619) 784-2749. E-mail: milligan @scripps.eduWe would like to thank M. Whittaker and B. Carragher for computational assistance. We would also like to thank D.P. Dias and M. Whittaker for critical reading of the manuscript, and for helpful discussions.
This work was supported by grants from the National Institutes of Health (AR39155 and AR44278) to R.A. Milligan. R.A. Milligan is an Established Investigator of the American Heart Association. J.D. Jontes is a predoctoral fellow of the Howard Hughes Medical Institute.
AM, actomyosin; BBM-I, brush border myosin-I; CaM, calmodulin; CTF, contrast transfer function; ELC, essential light chain; HC, heavy chain; LCBD, light chain-binding domain; 3D, three-dimensional.
1. | Bagshaw, C.R., and D.R. Trentham. 1974. The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction. Biochem. J. 141: 331-349 |
2. | Carragher, B.O., M. Whittaker, and R.A. Milligan. 1996. Helical processing using PHOELIX. J. Struct. Biol. 116: 107-112 |
3. | Cheney, R.E., and M.S. Mooseker. 1992. Unconventional myosins. Curr. Opin. Cell Biol. 4: 27-35 |
4. |
Collins, J.H., and
C.W. Borysenko.
1984.
The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase.
J.
Biol. Chem.
259:
14128-14135
|
5. | Collins, K., J.R. Sellers, and P. Matsudaira. 1990. Calmodulin dissociation regulates brush border myosin I (110-kD-calmodulin) mechanochemical activity. J. Cell Biol. 110: 1137-1147 [Abstract]. |
6. | Conzelman, K.A., and M.S. Mooseker. 1987. The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase. J. Cell Biol. 105: 313-324 [Abstract]. |
7. | Cooke, R.. 1986. The mechanism of muscle contraction. CRC Crit. Rev. Biochem. 21: 53-118 |
8. | Cooke, R., and E. Pate. 1985. The effects of ADP and phosphate on the contraction of muscle fibers. Biophys. J. 48: 789-798 [Abstract]. |
9. | Dantzig, J.A., Y.E. Goldman, N.C. Millar, J. Lacktis, and E. Homsher. 1992. Reversal of the cross-bridge force-generating transition by photogeneration of phosphate in rabbit psoas muscle fibres. J. Physiol. (Lond.). 451: 247-278 [Abstract]. |
10. | DeRosier, D.J., and P.B. Moore. 1970. Reconstruction of three-dimensional images from electron micrographs of structures with helical symmetry. J. Mol. Biol. 52: 355-369 |
11. | Drew, J.S., V.A. Harwalkar, and L.A. Stein. 1992. Product inhibition of the actomyosin subfragment-1 ATPase in skeletal, cardiac and smooth muscle. Circ. Res. 71: 1067-1077 [Abstract]. |
12. | Fersht, A.R. 1985. Enzyme structure and mechanism. W.H. Freeman and Co., New York. 121-154. |
13. | Garcia, A., E. Coudrier, J. Carboni, J. Anderson, J. Vandekerckhove, M. Mooseker, D. Louvard, and M. Arpin. 1989. Partial deduced sequence of the 110-kD calmodulin complex of the avian intestinal microvillus shows that this mechano-enzyme is a member of the myosin I family. J. Cell Biol. 109: 2895-2903 [Abstract]. |
14. | Gollub, J., C.R. Cremo, and R. Cooke. 1996. ADP release produces a rotation of the neck region of smooth myosin but not skeletal myosin. Nat. Struct. Biol. 3: 796-802 |
15. | Gutfreund, H. 1995. Kinetics for the Life Sciences. Cambridge University Press, Cambridge. 138-196. |
16. | Hai, C.M., and R.A. Murphy. 1989. Ca2+, cross-bridge phosphorylation and contraction. Annu. Rev. Physiol. 51: 285-298 |
17. | Halsall, D.J., and J.A. Hammer III.. 1990. A second isoform of chicken brush border myosin I contains a 29-residue inserted sequence that binds calmodulin. FEBS (Fed. Eur. Biochem. Soc.) Lett. 267: 126-130 . |
18. | Hammer, J.A. III.. 1994. The structure and function of unconventional myosins: a review. J. Muscle Res. Cell Motil. 15: 1-10 |
19. | Hayden, S.M., J.S. Wolenski, and M.S. Mooseker. 1990. Binding of brush border myosin I to phospholipid vesicles. J. Cell Biol. 111: 443-451 [Abstract]. |
20. | Hessler, D., S.J. Young, B.O. Carragher, M. Martone, J.E. Hinshaw, R.A. Milligan, E. Masliah, M. Whittaker, S. Lamont, and M.H. Ellisman. 1992. SYNU: software for visualization of 3-dimensional biological structures. In Microscopy: the Key Research Tool. C.E. Lyman, L.D. Peachey, and R.M. Fisher editors. 22:73-82., EMSA Inc., Milwaukee, WI. |
21. | Hibberd, M.G., J.A. Dantzig, D.R. Trentham, and Y.E. Goldman. 1985. Phosphate release and force generation in skeletal muscle fiberes. Science (Wash. DC). 228: 1317-1319 |
22. | Hill, T.L., and E. Eisenberg. 1981. Can free energy transduction be localized at some crucial part of the enzymatic cycle? Q. Rev. Biophys. 14: 463-511 |
23. | Homsher, E., J. Lacktis, and M. Regnier. 1997. Strain-dependent modulation of phosphate transients in rabbit skeletal muscle fibers. Biophys. J. 72: 1780-1791 [Abstract]. |
24. | Houdusse, A., M. Silver, and C. Cohen. 1996. A model of Ca2+-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch. Structure (Lond.). 4: 1475-1490 |
25. | Howe, C.L., and M.S. Mooseker. 1983. Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells. J. Cell Biol. 97: 974-985 [Abstract]. |
26. | Huxley, A.F.. 1957. Muscle structure and theories of contraction. Prog. Biophys. Biophys. Chem. 7: 255-318 . |
27. | Huxley, A.F., and R.M. Simmons. 1971. Proposed mechanism of force generation in striated muscle. Nature (Lond.). 233: 533-538 |
28. | Huxley, H.E.. 1969. The mechanism of muscle contraction. Science (Wash. DC). 164: 1356-1366 |
29. | Huxley, H.E., and M. Kress. 1985. Crossbridge behavior during muscle contraction. J. Muscle Res. Cell Motil. 6: 153-161 |
30. | Jones, T.A., J.Y. Zou, S.W. Cowan, and M. Kjeldgaard. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47: 110-119 |
31. | Jontes, J.D., and R.A. Milligan. 1997. Three-dimensional structure of brush border myosin-I at ~20 Å resolution by electron microscopy and image analysis. J. Mol. Biol. 266: 331-342 |
32. | Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. A 32° tail swing in brush border myosin I on ADP release. Nature (Lond.). 378: 751-753 |
33. | Ma, Y.-Z., and E.W. Taylor. 1994. Kinetic mechanism of myofibril ATPase. Biophys. J. 66: 1542-1553 [Abstract]. |
34. | Matsudaira, P.T., and D.R. Burgess. 1979. Identification and organization of the components in the isolated microvillus cytoskeleton. J. Cell Biol. 83: 667-673 [Abstract]. |
35. | Meador, W.E., A.R. Means, and F.A. Quiocho. 1992. Target enzyme recognition by calmodulin: 2.4Å structure of a calmodulin-peptide complex. Science (Wash. DC). 257: 1251-1255 |
36. | Millar, N.C., and E. Homsher. 1992. Kinetics of force generation and phosphate release in skinned rabbit soleus muscle fibres. Am. J. Physiol. 262: c1239-c1245 |
37. | Milligan, R.A., and P. Flicker. 1987. Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy. J. Cell Biol. 105: 29-39 [Abstract]. |
38. | Mooseker, M.S., and R.E. Cheney. 1995. Unconventional myosins. Annu. Rev. Cell Dev. Biol. 11: 633-765 . |
39. | Morgan, D.G., C. Owen, L.A. Melanson, and D.J. DeRosier. 1995. Structure of bacterial flagellar filaments at 11Å resolution: packing of the alpha helices. J. Mol. Biol. 249: 88-110 |
40. | Nishiye, E., A.V. Somlyo, K. Török, and A.P. Somlyo. 1993. The effects of MgADP on cross-bridge kinetics: a laser flash photolysis study of guinea-pig smooth muscle. J. Phyiol. 460: 247-271 [Abstract]. |
41. | Pollard, T.D., S.K. Doberstein, and H.G. Zot. 1991. Myosin-I. Annu. Rev. Physiol. 53: 653-681 |
42. | Rayment, I., W.R. Rypniewski, K. Schmidt-Base, R. Smith, D.R. Tomchick, M.M. Benning, D.A. Winkelmann, G. Wesenberg, and H.M. Holden. 1993a. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science (Wash. DC). 261: 50-58 |
43. | Rayment, I., H.M. Holden, M. Whittaker, C.B. Yohn, M. Lorenz, K.C. Holmes, and R.A. Milligan. 1993b. Structure of the actin-myosin complex and its implications for muscle contraction. Science (Wash. DC). 261: 58-65 |
44. |
Rosenfeld, S.S., and
E.W. Taylor.
1984.
The ATPase mechanism of skeletal and
smooth muscle acto-subfragment-1.
J. Biol. Chem.
259:
11908-11919
|
45. | Siemankowski, R.F., M.O. Wiseman, and H.D. White. 1985. ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proc. Natl. Acad. Sci. USA. 82: 658-662 [Abstract]. |
46. |
Sleep, J.A., and
R.L. Hutton.
1980.
Exchange between inorganic phosphate and
adenosine 5![]() |
47. |
Spudich, J.A., and
S. Watt.
1971.
The regulation of rabbit skeletal muscle contraction.
J. Biol. Chem.
246:
4866-4871
|
48. |
Swanljung-Collins, H., and
J.H. Collins.
1992.
Phosphorylation of brush border
myosin I by protein kinase C is regulated by Ca2+-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation.
J. Biol.
Chem.
267:
3445-3454
|
49. |
Taylor, E.W..
1991.
Kinetic studies on the association and dissociation of myosin
subfragment 1 and actin.
J. Biol. Chem.
266:
294-302
|
50. | Titus, M.A.. 1993. Myosins. Curr. Opin. Cell Biol. 5: 77-81 |
51. |
Trybus, K.M., and
E.W. Taylor.
1982.
Transient kinetics of adenosine 5![]() ![]() ![]() ![]() |
52. | Vibert, P., and C. Cohen. 1988. Domains, motions and regulation in the myosin head. J. Muscle Res. Cell Motil. 9: 296-305 |
53. | White, H.D., and E.W. Taylor. 1976. Energetics and mechanism of actomyosin ATPase. Biochemistry. 15: 5818-5826 |
54. | Whittaker, M., and R.A. Milligan. 1997. Conformational changes due to calcium-induced calmodulin dissociation in brush border myosin I-decorated F-actin revealed by cryoelectron microscopy and image analysis. J. Mol. Biol. 269: 548-557 |
55. | Whittaker, M., B.O. Carragher, and R.A. Milligan. 1995a. PHOELIX: a package for semi-automated helical reconstruction. Ultramicroscopy. 58: 245-259 |
56. | Whittaker, M., E.M. Wilson-Kubalek, J.E. Smith, L. Faust, R.A. Milligan, and H.L. Sweeney. 1995b. A 35Å movement of smooth muscle myosin on ADP release. Nature (Lond.). 378: 748-751 |
57. | Wolenski, J.S.. 1995. Regulation of calmodulin-binding myosins. Trends Cell Biol. 5: 310-316 . |
58. | Wolenski, J.S., S.M. Hayden, P. Forscher, and M.S. Mooseker. 1993. Calcium-calmodulin and regulation of brush border myosin-I MgATPase and mechanochemistry. J. Cell Biol. 122: 613-621 [Abstract]. |
59. | Zot, H.G.. 1995. Phospholipid membrane-associated brush border myosin-I activity. Cell. Motil. Cytoskeleton. 30: 26-37 |