Ca2+-induced activation of ATPase activity of myosin Va is accompanied with a large conformational change

We succeeded in expressing the recombinant full-length myosin Va (M5Full) and studied its regulation mechanism. The actin-activated ATPase activity of M5Full was significantly activated by Ca(2+), whereas the truncated myosin Va without C-terminal globular domain is not regulated by Ca(2+) and constitutively active. Sedimentation analysis showed that the sedimentation coefficient of M5Full undergoes a Ca(2+)-induced conformational transition from 14S to 11S. Electron microscopy revealed that at low ionic strength, M5Full showed an extended conformation in high Ca(2+) while it formed a folded shape in the presence of EGTA, in which the tail domain was folded back towards the head-neck region. Furthermore, we found that the motor domain of myosin Va folds back to the neck domain in Ca(2+) while the head-neck domain is more extended in EGTA. It is thought that the association of the motor domain to the neck inhibits the binding of the tail to the neck thus destabilizing a folded conformation in Ca(2+). This conformational transition is closely correlated to the actin-activated ATPase activity. These results suggest that the tail and neck domain play a role in the Ca(2+) dependent regulation of myosin Va.     

Ca2+-sensitive transition in the molecular conformation of molluscan muscle myosins

Filament assemblies of myosin molecules purified from scallop adductor muscles were stabilized by Ca2+ in the presence of ATP or ADP. Electron micrographs showed that the tail part of monomeric myosin molecules was folded in the absence of Ca2+, but was extended in the presence of Ca2+ at physiological ionic strength.     

Role of the tail in the regulated state of myosin 2

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

Myosin assembly critical for the enzyme activity of smooth muscle myosin phosphatase: effects of MgATP, ionic strength, and Mg(2+)

We suggested that an assembled form of phosphorylated myosin (P-myosin) might exhibit higher affinity for smooth muscle myosin phosphatase (SMMP) than dissociated P-myosin on the basis of the effect of MgATP [Sato and Ogawa (1999) J. Biochem. 126, 787-797]. To further deepen our understanding, we examined the SMMP activity and P-myosin assembly with various ionic strengths and Mg(2+) concentrations, with and without MgATP, all of which are well known to be critical for myosin assembly. The structure of myosin molecules was directly observed by electron microscopy using a rotary shadowing procedure, which was found to be consistent with the sedimentation assay. We found that the SMMP activity was always high when P-myosin was assembled. MgATP, which disassembled P-myosin mostly into a folded conformation, in contrast, decreased the enzyme activity. We also found that glycerol had a dissociating action on P-myosin, primarily dissociating it into an extended conformation, resulting in reduced SMMP activity, and that increases in the ionic strength and Mg(2+) (>5 mM) inhibited SMMP. These results indicate that myosin assembly is essential for SMMP activity.     

Regulation of the function of mammalian myosin and its conformational change

It has been known that the phosphorylation of the regulatory light chain, residing at the head/rod junction of the molecule activates the motor activity of smooth muscle and non-muscle conventional myosin (myosin II), and triggers a large conformational change of the molecule from the inhibited folded conformation to the active extended conformation. Recent structural analysis has revealed the structural basis of the inhibition of the motor function of the two heads in the inhibited conformation. On the other hand, recent studies have revealed that a processive unconventional myosin, myosin V, also shows a large change in the conformation from the folded to an extended form and this explains the activation mechanism of myosin V motor activity. These findings suggest the presence of a common scenario for the regulation of motor protein functions.     

Modification of thiols of gizzard myosin alters ATPase activity, stability of myosin filaments, and the 6-10 S conformational transition

The pattern of incorporation of [14C]N-ethylmaleimide (MalNEt) into gizzard myosin indicates the presence of two classes of thiols: rapidly and slowly modified. The first class contains two thiol residues, SH-A and SH-B, located in the myosin rod and the 17-kDa light chain, respectively, while the second contains at least two thiols located in the myosin heavy chain. Changes in ATPase activities upon modification occur rapidly or slowly, paralleling reaction of either the first or second class of thiols. Rapid changes include increases in the Ca2+- and Mg2+-activated activities of myosin alone, measured at ionic strengths below 0.3 M, and an increase and a decrease in the actin-activated activity of dephosphorylated and phosphorylated myosin, respectively. Modification of SH-A and SH-B with MalNEt is accompanied by stabilization of myosin filaments, seen as an increase in light-scattering intensity, and by destabilization of the folded, 10 S conformation of the myosin monomer. In the presence of 0.175 M NaCl and 1 mM MgATP, unmodified and MalNEt-modified myosin sediment in the ultracentrifuge as single components at 10.0 S and 6.0 S, respectively. The MalNEt-induced increase in the Ca2+- or Mg2+-activated ATPase activity, measured in the absence of actin, can be attributed either to stabilization of filaments or to destabilization of the 10 S conformation, depending on the ionic strength of the assay. Modification of the second class of thiols is accompanied by a decrease in K+-EDTA-activated activity and an increase in Ca2+-activated activity measured above 0.3 M NaCl, where myosin neither forms filaments nor assumes the 10 S conformation. These slow changes are characteristic of blocking the SH-1 thiols of skeletal-muscle myosin, but in gizzard myosin are attributable to modification of a less reactive thiol, SH-C.     

A conformational transition in gizzard heavy meromyosin involving the head-tail junction, resulting in changes in sedimentation coefficient, ATPase activity, and orientation of heads

Gizzard heavy meromyosin (HMM) sediments in the ultracentrifuge as a single peak, whose sedimentation coefficient (S20,w) decreases from 9 to 7.5 S upon increasing the NaCl concentration from 0.02 to 0.3 M. This decrease is accompanied by a parallel increase in Mg2+-ATPase activity, suggesting that both changes have a common molecular basis. Phosphorylation decreases S20,w and increases ATPase activity, while ATP increases S20,w. Sedimentation equilibrium studies indicate that HMM undergoes no detectable aggregation at 0.02 or 0.4 M NaCl, remaining monomeric with a molecular weight of 3.4 X 10(5). In contrast, S20,w of subfragment 1 does not change with changes in ionic strength, and its ATPase activity does not decrease at low ionic strengths. Electron micrographs of samples of HMM prepared at low ionic strength show that up to half of the molecules are flexed, i.e. the heads are bent at the neck and project back toward the tail, while the remaining molecules have either one or both of the heads pointing away from the tail. In samples prepared at high ionic strength only about 10% of the molecules are flexed. There is a linear relationship between the fraction of flexed molecules and S20,w, with no significant bending or folding of the tail and no detectable change in the shape of the heads. This correlation suggests that the changes in ATPase activity and S20,w may be a result of the reorientation of the heads.     

Correlation of conformation and phosphorylation and dephosphorylation of smooth muscle myosin

The rate of phosphorylation and dephosphorylation of smooth muscle myosin by myosin light chain kinase and by two myosin light chain phosphatases (gizzard phosphatase IV and aorta phosphatase) are measured in various conditions; the relationship between the rate of phosphorylation and dephosphorylation of myosin and the myosin conformation is also studied. The rate of dephosphorylation of myosin was completely inhibited in the presence of 1 mM MgCl2 and ATP at low ionic strength where phosphorylated myosin forms a folded conformation. The inhibition was released when myosin formed either an extended monomer or filaments. The rate of phosphorylation of myosin was also affected by the conformation of myosin. The rate for a folded myosin was slower than those for an extended monomer and filamentous myosin. The phosphorylation and dephosphorylation of heavy meromyosin, subfragment-1, and the isolated 20,000-dalton light chain are not inhibited at low ionic strength, and the rate of phosphorylation and dephosphorylation was decreased with increasing ionic strength. KCl dependence of the rate of phosphorylation and dephosphorylation of myosin was normalized by using KCl dependence of subfragment-1, and it was found that the marked inhibition of the rate of phosphorylation and dephosphorylation of myosin is closely related to the change from an extended to a folded conformation of myosin.     

Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation-related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded myosin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.     

Effects of Ca2+ on the conformation and enzymatic activity of smooth muscle myosin

The influence of Ca2+ on the enzymatic and physical properties of smooth muscle myosin was studied. The actin-activated ATPase activity of phosphorylated gizzard myosin and heavy meromyosin is higher in the presence of Ca2+ than in its absence, but this effect is found only at lower MgCl2 concentrations. As the MgCl2 concentration is increased, Ca2+ sensitivity is decreased. The concentration of Ca2+ necessary to activate ATPase activity is higher than that required to saturate calmodulin. The similarity of the pCa dependence of ATPase activity and of Ca2+ binding to myosin and the competition by Mg2+ indicate that these effects involved the Ca2+-Mg2+ binding sites of gizzard myosin. For the actin dependence of ATPase activity of phosphorylated myosin at low concentrations of MgCl2, both Vmax and Ka are influenced by Ca2+. The formation of small polymers by phosphorylated myosin in the presence of Ca2+ could account for the alteration in the affinity for actin. For the actin dependence of phosphorylated heavy meromyosin at low MgCl2 concentrations, Ca2+ induces only an increase in Vmax. To detect alterations in physical properties, two techniques were used: viscosity and limited papain hydrolysis. For dephosphorylated myosin, 6 S or 10 S, Ca2+-dependent effects are not detected using either technique. However, for phosphorylated myosin the decrease in viscosity corresponding to the 6 S to 10 S transition is shifted to lower KCl concentrations by the presence of Ca2+. In addition, a Ca2+ dependence of proteolysis rates is observed with phosphorylated myosin but only at low ionic strength, i.e. under conditions where myosin assumes the folded conformation.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

Ca2+-sensitive cross-bridge dissociation in the presence of magnesium pyrophosphate in skinned rabbit psoas fibers.

We find that at 6 degrees C in the presence of 4 mM MgPPi, at low or moderate ionic strength, skinned rabbit psoas fibers exhibit a stiffness and an equatorial x-ray diffraction pattern similar to that of rigor fibers. As the ionic strength is increased in the absence of Ca2+, both the stiffness and the equatorial x-ray diffraction pattern approach those of the relaxed state. This suggests that, as in solution, increasing ionic strength weakens the affinity of myosin cross-bridges for actin, which results in a decrease in the number of cross-bridges attached. The effect is Ca2+-sensitive. Assuming that stiffness is a measure of the number of cross-bridge heads attached, in the absence of Ca2+, the fraction of attached cross-bridge heads varies from approximately 75% to approximately 25% over an ionic strength range where ionic strength in solution weakens the binding constant for myosin subfragment-1 binding to unregulated actin by less than a factor of 3. Therefore, this phenomenon appears similar to the cooperative Ca2+-sensitive binding of S1 to regulated actin in solution (Greene, L. E., and E. Eisenberg, 1980, Proc. Natl. Acad. Sci. USA, 77:2616). By comparing the binding constants in solution and in the fiber under similar conditions, we find that the "effective actin concentration," that is, the concentration that gives the same fraction of S1 molecules bound to actin in solution as cross-bridge heads are bound to actin in a fiber, is in the millimolar range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinesin undergoes a 9 S to 6 S conformational transition.

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.     

Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.     

Truncation of vertebrate striated muscle myosin light chains disturbs calcium-induced structural transitions in synthetic myosin filaments

Electron microscopy and negative staining techniques have been used to show that the proteolytic removal of 13 amino acids from the N-terminus of essential light chain 1 and 19 amino acids from the N-terminus of the regulatory light chain of rabbit skeletal and cardiac muscle myosins destroys Ca(2+)-induced reversible movement of subfragment-2 (S2) with heads (S1) away from the backbone of synthetic myosin filaments observed for control assemblies of the myosin under near physiological conditions. This is the direct demonstration of the contribution of the S2 movement to the Ca(2+)-sensitive structural behavior of rabbit cardiac and skeletal myosin filaments and of the necessity of intact light chains for this movement. In muscle, such a mobility might play an important role in proper functioning of the myosin filaments. The impairment of the Ca(2+)-dependent structural behavior of S2 with S1 on the surface of the synthetic myosin filaments observed by us may be of direct relevance to some cardiomyopathies, which are accompanied by proteolytic breakdown or dissociation of myosin light chains.     

Ca2+ and ionic strength dependencies of S1-ADP binding to actin-tropomyosin-troponin: regulatory implications

Skeletal and cardiac muscle contraction are inhibited by the actin-associated complex of tropomyosin-troponin. Binding of Ca(2+) to troponin or binding of ATP-free myosin to actin reverses this inhibition. Ca(2+) and ATP-free myosin stabilize different tropomyosin-actin structural arrangements. The position of tropomyosin on actin affects the binding of ATP-free myosin to actin but does not greatly affect myosin-ATP binding. Ca(2+) and ATP-free myosin alter both the affinity of ATP-free myosin for actin and the kinetics of that binding. A parallel pathway model of regulation simulated the effects of Ca(2+) and ATP-free myosin binding on both equilibrium binding of myosin-nucleotide complexes to actin and the general features of ATPase activity. That model was recently shown to simulate the kinetics of myosin-S1 binding but the analysis was limited to a single condition because of the limited data available. We have now measured equilibrium binding and binding kinetics of myosin-S1-ADP to actin at a series of ionic strengths and free Ca(2+) concentrations. The parallel pathway model of regulation is consistent with those data. In that model the interaction between adjacent regulatory complexes fully saturated with Ca(2+) was destabilized and the inactive state of actin was stabilized at high ionic strength. These changes explain the previously observed change in binding kinetics with increasing ionic strength.     

Globular tail of myosin-V is bound to vamp/synaptobrevin

VAMP/synaptobrevin is one of a number of v-SNAREs involved in vesicular fusion events in neurons. In a previous report, VAMP was shown to form a complex with synaptophysin and myosin V, a motor protein based on the F-actin, and that myosin V was then released from the complex in a Ca(2+)-dependent manner. Here, we found that VAMP alone is bound to myosin V in a Ca(2+)-independent manner, and determined that the globular tail domain of myosin V is its binding site. The syntaxin-VAMP-myosin V formed in the presence of Ca(2+)/calmodulin (CaM). In the absence of CaM, only syntaxin-VAMP, or VAMP-myosin V complex was formed. Our results suggest that VAMP acts as a myosin V receptor on the vesicles and regulates formation of the complex.     

Conformations of vertebrate striated muscle myosin monomers in equilibrium with filaments.

Porcine cardiac myosin monomers in equilibrium with filaments under physiological conditions were observed to have two conformations, extended and folded forms, upon electron microscopy and gel filtration HPLC. The conformational state was independent of ATP and the phosphorylation of regulatory light chain. The folded monomers of cardiac myosin were mainly in an open conformation with only one bend in the tail, and may not trap the hydrolysis products of ATP, as assessed by single turnover experiments. These properties are similar to those of the folded monomers of rabbit skeletal myosin [Katoh, T., Konishi, K., and Yazawa, M. (1998) J. Biol. Chem. 273, 11436-11439]. The conformational states of skeletal and cardiac myosin monomers were not affected by pH between 7.0 and 8.5. Although significant disassembly of filaments and thus an increase in the monomer concentration were observed with an increase in pH. The results indicate that the pH-dependent change in filament assembly is due to a shift of equilibrium between the filaments and extended monomers toward filament disassembly. The Mg2+-ATPase activity of these myosin monomers decreased with a decrease in the salt concentration below approximately 0.1 M, suggestive of the formation of a closed conformation similar to the conformation of 10S smooth myosin. The results suggest that the conformational change from the extended to the folded form is a common property of various myosin IIs.     

Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification

A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.