(Received for publication, October 3, 1994)
From the
Recent structural evidence (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A.(1993) Science 261, 58-65) suggests that the two heads of skeletal muscle myosin interact when the protein is bound to filamentous actin. Direct chemical cross-linking experiments show that the two heads of smooth muscle myosin interact in the presence of filamentous actin and the absence of ATP (Onishi, H., Maita, T., Matsuda, G., and Fujiwara, K.(1992) Biochemistry 31, 1201-1210). Head-head interactions may be important in the mechanism of phosphorylation-dependent regulation of smooth muscle myosin. To explore the structural elements essential for phosphorylation-dependent regulation, we purified a proteolytic fragment of chicken gizzard myosin containing only one head attached to an intact tail. This molecule contained a partially digested regulatory light chain, which was replaced with exogenously added intact light chain in either the thiophosphorylated or the unphosphorylated state. Control experiments showed that this replacement was nearly quantitative and did not alter the actin-activated ATPase of this myosin. Electron micrographs confirmed that the single-headed preparation contained an intact form of single-headed myosin. The unphosphorylated single-headed myosin hydrolyzed ATP rapidly and moved actin filaments in an in vitro motility assay. Phosphorylation had minimal effects upon these properties. Therefore, we conclude that phosphorylation-dependent regulation in this myosin requires two heads. These findings may have important implications in studies of other regulated motor proteins that contain two motor domains.
Smooth muscle contraction is initiated by phosphorylation of
myosin's regulatory light chain
(RLC)()(1, 2, 3) . In
vitro, this myosin and similar myosin IIs (4) must be
phosphorylated to hydrolyze ATP rapidly in the presence of actin and to
move actin filaments. Phosphorylation appears to be sufficient to
activate smooth muscle myosin, independent of changes in filament
assembly(5) . Heavy meromyosin, having two heads but lacking
the carboxyl-terminal two-thirds of the tail, is regulated by
phosphorylation(6, 7) . Subfragment-1, containing a
single head and no tail, is always active (7, 8) .
These data suggest that the ``off'' state requires head-head
or head-tail interactions. To differentiate between these two
possibilities, we have investigated a highly purified single-headed
proteolytic fragment isolated from a proteolytic digest of smooth
muscle myosin.
Single-headed proteolytic fragments of myosins from various muscles that exhibit different types of regulation have been previously studied. Purified single-headed fragments of unregulated skeletal muscle myosin have been shown to assemble into normal filaments(9, 10, 11) and produce normal isometric tension per head(11, 12) . Cooperative interaction between the heads is not essential to develop sliding force (11) .
To our knowledge, there are no reports of regulatory
studies with purified single-headed fragments of myosins that exhibit
myosin-linked regulation. However, the question of the role of two
heads in regulation has been previously addressed. Scallop S1 is
similar to smooth S1 in that it is unregulated(13) . In studies
with unpurified protease digestion mixtures of scallop-striated muscle
myosin, the single-headed myosin fragment appears to retain regulation (13) by Ca binding to the interface between
the regulatory and essential light chains(14) . Similar
experiments with unpurified smooth muscle myosin digests suggest that
the single-headed myosin lacks phosphorylation-dependent
regulation(15) . This result contradicted an earlier report of
the insoluble fraction of papain digests of smooth muscle myosin,
suggesting that the single-headed form required phosphorylation for
actin-activated ATPase(16) . Thus, from these initial studies
with unpurified single-headed fragments, it is not clear whether the
structural interactions responsible for the two myosin-linked
regulatory mechanisms, Ca
binding and
phosphorylation, are fundamentally different or whether they might be
similar.
In our studies of proteolytic digestion of smooth muscle myosin, we found that the single-headed form is a transient intermediate that is never present in large amounts relative to the total number of myosin heads in a mixture. For this reason, it is difficult to study the properties of the single-headed form without further purification. Our approach here was to avoid potential problems of proteolytic digestion mixtures and to first highly purify a single-headed fragment of smooth muscle myosin in preparative amounts. In this way, the single-headed myosin was not contaminated with large amounts of double-headed myosin and S1, normally produced in a proteolytic digest. Isolation of a single-headed fragment in a purified form has allowed an investigation of the phosphorylation-dependent regulatory behavior by steady-state ATPase and in vitro motility measurements.
Myosin was purified from frozen chicken gizzards (17) obtained from Pell-Freeze. The preparative isolation of
single-headed myosin combines aspects from previous single-headed
myosin
preparations(10, 12, 13, 18) . The
pH of all solutions was adjusted at 4 °C. All dialyses and
centrifugations were at 4 °C. Freshly purified myosin (400 mg) was
digested in filamentous form at 4 mg/ml in 0.2 M ammonium
acetate, pH 7.8, 2 mM EGTA, 1 mM DTT with 2 µg/ml
papain (Sigma, 20 units/mg protein) for 27 min at 25 °C. After
quenching the digestion with 5 µg/ml leupeptin, the soluble heads
(S1) were removed by twice centrifuging and washing the myosin
filaments with digestion buffer including leupeptin. The resulting
pellet, containing double-headed and single-headed myosin and rods, was
dialyzed overnight into actin binding buffer (0.35 M NaCl, 10
mM sodium phosphate, pH 7.2, 1 mM MgCl, 1
mM EGTA, 5 mM DTT) and centrifuged (350,000
g, 30 min) to remove insoluble material. F-actin from rabbit
skeletal muscle (19) was dialyzed extensively versus 50 mM KCl, 1 mM MgCl
, 0.2 mM DTT, 10 mM Tris, pH 8.2, to remove ATP. The actin and the
digested myosin were then mixed in a ratio of 0.5 mg of actin to
1
mg of digest (estimated using
=
5.6 cm
). The final concentrations were 4-5
mg/ml myosin digest and 2-2.5 mg/ml actin. After adjusting the
final KCl concentration to 0.3 M, the mixture was centrifuged
(170,000
g, 50 min), leaving myosin rods in the
supernatant. The pellet was homogenized in actin binding buffer and
centrifuged twice more to completely remove rods from the pellet.
Single-headed myosin was selectively released from the pellet by
homogenizing in actin binding buffer containing 3 mM sodium
pyrophosphate and centrifuging as before. Double-headed myosin and
actin remained in the pellet. The yield of single-headed myosin
(
= 4.3
cm
(12) ) could be doubled by repeating the
last resuspension and centrifugation. The purification procedure was
monitored by nondenaturing gel electrophoresis in the presence of
sodium pyrophosphate(20) . Single-head myosin was identified as
an intermediate band that transiently appeared in papain digestion time
courses, as in the analysis of scallop myosin digests(13) .
Gels were scanned with a BioImage Visage 60-110 scanner to
estimate purity. The clipped RLC of the single-headed myosin was
replaced with exogenously added RLC or tpRLC(21) . Pellets were
then resuspended in 15 mM Tris, pH 7.5, at 25 °C, 0.3 M KCl, 5 mM MgCl
, 1.0 mM EGTA,
0.1 mM DTT, and the solution was centrifuged to remove
insoluble material. A single-headed sample was prepared that was
treated identically, except that light chains were not added and the
sample remained at 4 °C during the exchange step. A parallel set of
double-headed samples was also prepared. Complete exchange was verified
by analyzing the samples on a 4-15% polyacrylamide SDS gel (see Fig. 1B) or on a polyacrylamide gel in the presence of
urea (21) (data not shown). Electron microscopy was performed
on a Hitashi 600 electron microscope operated at 75 kV. Specimens were
prepared (22) at 22 µg/ml protein in 66% glycerol, 0.5 M KCl, 20 mM Tris, pH 8.2, 10 mM DTT, 1
mM EGTA.
Figure 1:
Characterization of single-headed
myosin. A, nondenaturing gel electrophoresis. dh,
double-headed; sh, single-headed. Lane1,
initial double-headed myosin (7 µg); lane2,
after papain digestion and S1 removal (5 µg); lane3, single-headed myosin preparation (3 µg); lane4, same as lane3 except 4.2 µg; lane5, rod fraction (5 µg). B, the
extent of exchange of exogenously added RLC into single-headed myosin
(and double-headed myosin controls) was monitored by SDS-gel
electrophoresis. Lane1, initial double-headed
myosin; lane2, after papain digestion and S1
removal; lane3, double-headed undigested control
prior RLC exchange; lane4, double-headed undigested
control after RLC exchange; lane5, double-headed
undigested control after tpRLC exchange; lane6,
single-headed myosin prior to adding RLC; lane7,
single-headed myosin after RLC exchange; lane8,
single-headed myosin after tpRLC exchange. C,
platinum-shadowed carbon-coated images of double-headed myosin; D, single-headed myosin exchanged with RLC (samples shown in lanes4 and 7 in B, respectively). Bar = 50
nm.
The in vitro motility assays were
performed, and the data were analyzed as described (23) except
the motility buffer contained 60 mM KCl, 20 mM MOPS
(pH 7.2), 5 mM MgCl, 20 mM DTT, 0.1
mM EGTA, 200 nM gizzard tropomyosin, 2.5 mg/ml
glucose, 0.1 mg/ml glucose oxidase, 2 µg/ml catalase, 0.7%
methylcellulose, and the assay temperature was 30 °C.
Our approach was to isolate in preparative amounts and
subsequently characterize a single-headed fragment isolated from a
papain digestion of gizzard myosin. Our single-headed myosin
preparation contained 95-96% single-headed myosin and 4-5%
double-headed myosin as determined by densitometric scanning of a
non-denaturing gel (Fig. 1A, lanes3 and 4). SDS-gel electrophoresis of the single-headed
preparation (Fig. 1B, lane6) showed
that some of the heavy chains (hc, 200 kDa) were clipped
by papain at the junction between the 50- and 20-kDa S1 head segments
to generate the carboxyl-terminal
140-kDa and amino-terminal
70-kDa fragments (18, 24) . The
120-kDa rod
fragment verifies the presence of single-headed myosin. Approximately
80% of the RLC, clipped between Arg-16 and Thr-17, migrated just below
the essential light chain (Fig. 1B, lane6) and could not be phosphorylated by myosin light chain
kinase(25) . Therefore, the endogenous clipped RLC was
exchanged (21) with exogenously added RLC (Fig. 1B, lane7) or tpRLC (Fig. 1B, lane8). Similar RLC
exchange procedures have been used by other investigators who have
shown that this method does not significantly alter the ATPase activity
and the ability of the myosin to undergo the salt-dependent transition
between 6 S and 10 S forms(26, 27) . Gel scanning
showed that both double-headed (Fig. 1B, lanes4 and 5) and single-headed (lanes7 and 8) exchanged samples contained the same ratio of
native RLC to essential light chain as untreated native myosin (lane1). Electron micrographs showed an intact
single-headed myosin (Fig. 1D), which clearly lacks a
head domain upon comparison with double-headed myosin (Fig. 1C). Several fields of view were analyzed (data
not shown), and this analysis showed that only 1-2% of the myosin
in our single-headed preparation was double-headed.
The MgATPase activities of both untreated and RLC-exchanged double-headed myosins were activated with actin upon thiophosphorylation by about a factor of 40 (Table 1). In contrast, single-headed myosins, with either clipped or intact RLC, had high MgATPase activity with actin even without thiophosphorylation. Thiophosphorylation increased this activity only by a factor of about 1.6. These data are consistent with a lack of phosphorylation-dependent regulation in the single-headed myosin.
We measured in vitro actin filament movement as another test for phosphorylation-dependent regulation (Fig. 2). The thiophosphorylated double-headed myosin moved actin filaments at about 1.4 µm/s (Fig. 2A), whereas unphosphorylated double-headed myosin did not move at a significant rate (Fig. 2B). Single-headed myosin did not show this phosphorylation-dependent regulation of actin filament movement (Fig. 2, C and D). The mean velocities in µm/s of the actin filaments that were consistently moving (i.e. where the ratio of the standard deviation to the mean is less than 0.3) (23) were 1.5 ± 0.3 for A, 0.13 ± 0.12 for B, 0.95 ± 0.31 for C, and 0.65 ± 0.24 for D. The mean rate of movement for single-headed myosin with tpRLC was similar to the mean rate for unphosphorylated single-head myosin. The percentage of actin filaments that were moving was high in the case of single-headed myosin regardless of thiophosphorylation (Fig. 2, C and D). These data suggest that two heads are required for phosphorylation-dependent regulation of actin filament movement.
Figure 2: In vitro motility assays of myosins. All samples were exchanged with indicated RLC and treated as described for the samples in Table 1. The histograms show the velocity of all actin filaments observed in the field of view. A, thiophosphorylated double-headed myosin; B, unphosphorylated double-headed myosin; C, thiophosphorylated single-headed myosin; D, unphosphorylated single-headed myosin. The blackbars represent the velocities that are indistinguishable from the apparent velocity of filaments that are not moving (i.e. filaments bound to myosin heads in the absence of ATP(40) ). For A, these data were similar to data collected for samples that were phosphorylated during the motility assay on the coverslip.
The
single-headed myosin could lack regulation because it contains
papain-cleaved heavy chains (Fig. 1B), even though
double-headed species with similarly cleaved chains by Staphylococcus aureus protease(7) ,
chymotrypsin(6) , or myopathic hamster protease (28) are known to be well regulated. To address this point, we
prepared 95% purified single-headed gizzard myosin (data not shown) by
digesting with S. aureus protease (15) . Neither the S.aureus- nor the papain-prepared single-headed
myosins showed evidence for a subpopulation of regulated molecules in
single turnover experiments (29) using formycin triphosphate. ()Thus, the unregulated nature of single-headed myosin is
most readily attributed to the absence of one head.
These data show that an interaction between the two heads, rather than interaction of the head with the tail, is critical for the ``off'' state of unphosphorylated smooth muscle myosin. Phosphorylation at Ser-19 of the RLC relieves this inhibitory interaction, allowing the myosin to adopt force-generating conformations in the presence of actin. The lack of head-head interaction can explain why isolated myosin heads (S1) are active in both the phosphorylated and unphosphorylated states(7, 8) . Furthermore, as expected for molecules capable of head-head interaction, all double-headed proteolytic fragments studied to date have been found to be regulated(6, 7, 30) .
By analogy with the three-dimensional structure of skeletal myosin heads (31) and a fragment of the scallop myosin head(14) , Ser-19 of the smooth muscle myosin RLC is within a disordered amino terminus that may bind to portions of the myosin structure that were not visualized in the two crystal structures(31) . This binding region could be the coiled-coil S2 region or the other head, via either the heavy chain or the light chain regions. Phosphorylation probably effects the conformation of the amino terminus of the RLC as phosphorylation protects against proteolysis(32) . Perhaps upon phosphorylation of the RLC within a myosin molecule, elements of this amino-terminal portion of the light chain destabilize the interaction between the heads, probably by affecting the conformation at the hinge region (33) between the head and the tail. Residues 13-16 of the RLC of gizzard myosin appear to fit the requirements of such an element. Without these residues, the actin-activated ATPase remains inhibited regardless of phosphorylation. Furthermore, regulated and unregulated vertebrate myosin RLCs do not share homology in this region(34) . Although the exact mechanism of action of this amino-terminal portion of the light chain is not clear, the COOH-terminal portion of the RLC is likely to be an essential element(27) .
It has been shown that phosphorylation of both heads must occur before the actomyosin ATPase is fully activated(35, 36, 37) . In addition, dephosphorylation of one head appears to be sufficient to deactivate the whole molecule in both the filamentous and monomeric states(37) . In light of the importance of head-head interaction in the unphosphorylated state, it appears that in singly phosphorylated myosin, the phosphorylated head remains inactive because the other unphosphorylated head still retains elements of interaction with the phosphorylated head, thus restricting it from adopting force-generating conformations in the presence of actin. Upon double phosphorylation, both heads would become free, and the entire molecule could adopt the fully active conformation.
When bound to actin in
the absence of ATP, the two heads of unphosphorylated smooth muscle
heavy meromyosin interact asymmetrically between Lys-65, within a
-barrel extending away from the bulk of the head(38) , and
Glu-168 on the opposite side of the head(38, 39) .
Interestingly, the amino acids corresponding to the
-barrel are
missing from unconventional myosin Is, which are single-headed and do
not form filaments. This
-barrel may be a critical domain, which
is involved in the intramolecular head-head interactions that we
propose are regulated by phosphorylation in smooth muscle myosin.