Shape and flexibility of the myosin molecule

Molecules of rabbit skeletal myosin have been examined in the electron microscope after drying at low temperature from solutions containing ethylene glycol or glycerol and rotary-shadowing with platinum. Analysis of the structure has been assisted by stereo-photography. While the general appearance, two heads attached to a long tail, is similar to that described by Slayter & Lowey (1967), more detail about the shape and size of the heads can be discerned and new information has been obtained about the flexibility of the tail and the head-tail junction.The heads are 190 Å long and wider at their ends than near the junction with the tail; the shape resembles that of a pear. The length is appreciably greater than the generally accepted value for subfragment 1, the proteolytic fragment of myosin. The heads are flexibly attached to the tail and can assume a wide range of tilt angles.Because the point where the two heads join the tail can be identified, the length of the tail, 1560 (±50) Å, can be measured more accurately than formerly. While all parts of the tail are somewhat flexible, sharp bends often occur at a well-defined site 430 Å from the head-tail junction. The demonstration of hinges at the head-tail junction and in the tail provides strong support for H. E. Huxley's (1969) hypothesis for the mechanism of muscle contraction.     

丝瓜肌球蛋白的电子显微学研究

从丝瓜 (Luffacylindrica (L .)Roem .)卷须中纯化得到分子量为 174kD的肌球蛋白 ,并对其进行了酶学与电子显微学的研究。这种肌球蛋白具有肌动蛋白激活的MgATPase活性 ,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别。电子显微学研究表明 :它有两个头部 (大小和形状与动物肌肉的肌球蛋白相似 )和一条相对较短的尾部。还对丝瓜卷须的肌动蛋白进行了观测 ,偶尔发现一些尾部有球状结构的肌球蛋白。该肌球蛋白的免疫特性和超微结构证明了它由 2条重链组成 ,并与传统的肌球蛋白相似。然而 ,这种 174kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究。     

Movement of myosin fragments in vitro: domains involved in force production

We have used the Nitella-based movement assay to localize the site of force production in myosin. Methods were developed to use nonfilamentous myosin or proteolytic fragments of myosin in place of the thick filaments used in the original assay. In the experiments described here, the tail of myosin or its subfragments is anchored via antibodies to the surface of small particles. Nonfilamentous myosin or its subfragments move along Nitella actin cables at speeds similar to those obtained with filamentous myosin. We generated short HMM, a myosin fragment containing the heads and only 400 A of the tail. Although short HMM lacks the "hinge" region proposed by Harrington to be the site of force generation, and is incapable of forming thick filaments, it moves along actin at speeds above 1 micron/sec. Therefore, neither a thick filament nor the carboxy-terminal 1100 A of the tail is required for movement along actin. The results indicate that force production occurs in or near the myosin heads.     

丝瓜肌球蛋白的电子显微学研究

从丝瓜(Luffa cylindrica (L.) Roem.)卷须中纯化得到分子量为174kD的肌球蛋白,并对其进行了酶学与电子显微学的研究.这种肌球蛋白具有肌动蛋白激活的MgATPase活性,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别.电子显微学研究表明:它有两个头部(大小和形状与动物肌肉的肌球蛋白相似)和一条相对较短的尾部.还对丝瓜卷须的肌动蛋白进行了观测,偶尔发现一些尾部有球状结构的肌球蛋白.该肌球蛋白的免疫特性和超微结构证明了它由2条重链组成,并与传统的肌球蛋白相似.然而,这种174 kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究.     

Unshadowed myosin molecules: STEM mass-maps of myosin heads.

Myosin molecules were directly visualized without heavy metal shadowing by scanning transmission electron microscopy (STEM) under low dose conditions. The general appearance and dimensions of heavy metal-free molecules were similar to those of shadowed myosin, either after freeze-drying without or air-drying with glycerol. Two characteristic configurations of myosin head regions were found, a first type showing two pear-shaped heads with narrow necks and a second type showing two heads connected by an extra mass in the central regulatory domain where the light chains are located. The mass of the latter type (mol. wt. = 265 +/- 39 kd) is in excellent accordance with biochemical data whereas the mass of the first type is somewhat lower (mol. wt. 219 +/- 44 kd).     

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.     

Substructure of the myosin molecule. 3. Preparation of single-headed derivatives of myosin

Native myosin has two globular regions attached to an a-helical rod. Papain is able to cleave the globular “heads” from the rod, leading to the formation of a variety of single-headed molecules. Among these subfragments are isolated globules (HMM S-1) and single globules attached to helical rods of lengths varying from 500 to 1400 Å. These subfragments can be separated from the other products of the proteolytic digestion by salt elution from a DEAE-cellulose column. Some of the properties of single-headed heavy meromyosin and myosin have been determined by hydrodynamic methods, and shadow-cast preparations of these subfragments have been directly visualized by electron microscopy. In addition to providing further evidence for the presence of two similar halves in myosin, these new subfragments can be used in studies related to the question of why myosin has two active “heads”.     

Negative staining of myosin molecules

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.     

Three-dimensional structure of myosin subfragment-1 from electron microscopy of sectioned crystals

Image analysis of electron micrographs of thin-sectioned myosin subfragment-1 (S1) crystals has been used to determine the structure of the myosin head at approximately 25-A resolution. Previous work established that the unit cell of type I crystals of myosin S1 contains eight molecules arranged with orthorhombic space group symmetry P212121 and provided preliminary information on the size and shape of the myosin head (Winkelmann, D. A., H. Mekeel, and I. Rayment. 1985. J. Mol. Biol. 181:487-501). We have applied a systematic method of data collection by electron microscopy to reconstruct the three-dimensional (3D) structure of the S1 crystal lattice. Electron micrographs of thin sections were recorded at angles of up to 50 degrees by tilting the sections about the two orthogonal unit cell axes in sections cut perpendicular to the three major crystallographic axes. The data from six separate tilt series were merged to form a complete data set for 3D reconstruction. This approach has yielded an electron density map of the unit cell of the S1 crystals of sufficient detail. to delineate the molecular envelope of the myosin head. Myosin S1 has a tadpole-shaped molecular envelope that is very similar in appearance to the pear-shaped myosin heads observed by electron microscopy of rotary-shadowed and negatively stained myosin. The molecule is divided into essentially three morphological domains: a large domain on one end of the molecule corresponding to approximately 60% of the total molecular volume, a smaller central domain of approximately 30% of the volume that is separated from the larger domain by a cleft on one side of the molecule, and the smallest domain corresponding to a thin tail-like region containing approximately 10% of the volume. This molecular organization supports models of force generation by myosin which invoke conformational mobility at interdomain junctions within the head.     

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.     

Electric birefringence study of the solution structure of chymotrypsin-cleaved Acanthamoeba myosin II

After removal of the 66 COOH-terminal amino acids from each of its two heavy chains by chymotrypsin digestion, Acanthamoeba myosin II forms only parallel dimers under conditions in which native myosin II forms bipolar filaments (Kuznicki, J., Cote, G. P., Bowers, B., and Korn, E. D. (1985) J. Biol. Chem. 260, 1967-1972). We have studied the solution structure of the chymotrypsin-cleaved myosin II by electric birefringence. Only two species, known to be monomer and parallel dimer from previous studies, were detected. The contribution to the birefringence decay from dimer increased from about 10 to 70% as the KCl concentration was lowered from 100 mM to 0 in 50% glycerol. At all ionic strengths, the monomer had a relaxation time corrected to water at 20 degrees C of 8.2 microseconds, whereas a relaxation time of 10.3 microseconds was expected for monomers with straight rigid rods. This strongly indicates that the myosin rod in solution is bent. On the assumption that there is a single bend 26 nm from the tip of the tail, as suggested by electron microscopy, it was calculated that the average bend angle would be 110 degrees, in solution, if as seems most likely, the average angle between the two globular heads were 180 degrees. The observed relaxation time of the dimer corrected to water at 20 degrees C was 25 microseconds, independent of ionic strength, which, if the motion of the heads were unrestricted, is consistent with a structure for a parallel dimer in which either the two monomer subunits have straight rigid rods and are staggered by about 28 nm or only one is bent and the stagger is 30 nm. As described in the accompanying Appendix, either of these dimers can be assembled into a bipolar filament compatible with the apparent structure of filaments of native myosin II (Pollard, T.D. (1982) J. Cell Biol. 95, 816-825).     

Proteolytic substructure of brain myosin

Individual bovine brain myosin molecules visualized by electron microscopy consist of two globular heads and a fibrous tail, like myosin molecules from other sources. Brain myosin, however, showed much lower solubility at moderate to high ionic strength (0.2 to 0.4 M KCl) than gizzard myosin, and the filaments formed at low ionic strength in the presence of Mg2+ were fairly resistant to low concentrations of ATP, by which gizzard myosin filaments were completely solubilized. Brain myosin was digested with low concentrations of papain, alpha-chymotrypsin, or trypsin, and the fragmentation patterns were analyzed by means of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sedimentation at low ionic strength, and electron microscopy of the fragments produced. The results indicate that all of the proteases cleave the myosin molecule primarily at sites located in the neck or in the head close to the neck, suggesting that the brain myosin molecule contains a hinge region or an open peptide stretch around these sites. The differences as well as the similarities between the proteolytic fragmentation patterns of brain myosin and other myosins are discussed.     

Myosin molecule packing within the vertebrate skeletal muscle thick filaments. A complete bipolar model

Computer modelling related to the real dimensions of both the whole filament and the myosin molecule subfragments has revealed two alternative modes for myosin molecule packing which lead to the head disposition similar to that observed by EM on the surface of the cross-bridge zone of the relaxed vertebrate skeletal muscle thick filaments. One of the modes has been known for three decades and is usually incorporated into the so-called three-stranded model. The new mode differs from the former one in two aspects: (1) myosin heads are grouped into asymmetrical cross-bridge crowns instead of symmetrical ones; (2) not the whole myosin tail, but only a 43-nm C-terminus of each of them is straightened and near-parallel to the filament axis, the rest of the tail is twisted. Concurrent exploration of these alternative modes has revealed their influence on the filament features. The parameter values for the filament models as well as for the building units depicting the myosin molecule subfragments are verified by experimental data found in the literature. On the basis of the new mode for myosin molecule packing a complete bipolar structure of the thick filament is created.     

Skip residues correlate with bends in the myosin tail

Sharp bends have previously been observed in the tail of the skeletal myosin molecule at well-defined positions 44, 75 and 135 nm from the head-tail junction, and in vertebrate smooth myosin at two positions about 45 and 96 nm from this junction. The amino acid sequence of the heavy chain does not straightforwardly account for such bending on the original model of the tail in which an invariant proline residue is present at the head-tail junction and the repeating seven amino acid pattern of hydrophobic residues lies entirely in the tail. Recently, a revised model has been proposed by Rimm et al. in which the first seven to eight heptads lie in the heads. It is shown here that with this model the observed bends in the tail of skeletal myosin coincide with three of the four additional (skip) residues that interrupt the heptad repeat. It is concluded that the skip residues, by causing localized instability of the coiled-coil, are responsible for the bends. Smooth myosin lacks the second of these skip residues explaining the absence of a bend at 75 nm.     

MgATP binding changes neither the shape nor the flexibility of the heads of single myosin molecules.

The structural mechanism by which myosin heads exert force is unknown. One possibility is that the tight binding of the heads to actin drives them into a force-generating configuration. Another possibility is that the force-generating conformational change is inherent to the myosin heads. In this case the heads would make force by changing their shape according to the species of nucleotide in their active sites, the tight attachment to actin serving only to provide traction. To test this latter possibility, we used negative stain electron microscopy to search for a MgATP-induced shape change in the heads of single myosin molecules. We compared the heads of 10S smooth muscle myosin monomers (wherein MgATP is trapped at the active site) with the MgATP-free heads of 6S monomers. We found that to a resolution of about 2 nm, MgATP binding to the unrestrained myosin head does not drive it to change its shape or its flexibility. This result suggests that the head makes force by virtue of an induced fit to actin.     

Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.     

Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site

The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.     

Direct localization of monoclonal antibody-binding sites on Acanthamoeba myosin-II and inhibition of filament formation by antibodies that bind to specific sites on the myosin-II tail

Electron microscopy of myosin-II molecules and filaments reacted with monoclonal antibodies demonstrates directly where the antibodies bind and shows that certain antibodies can inhibit the polymerization of myosin-II into filaments. The binding sites of seven of 23 different monoclonal antibodies were localized by platinum shadowing of myosin monomer-antibody complexes. The antibodies bind to a variety of sites on the myosin-II molecule, including the heads, the proximal end of the tail near the junction of the heads and tail, and the tip of the tail. The binding sites of eight of the 23 antibodies were also localized on myosin filaments by negative staining. Antibodies that bind to either the myosin heads or to the proximal end of the tail decorate the ends of the bipolar filaments. Some of the antibodies that bind to the tip of the myosin-II tail decorate the bare zone of the myosin-II thin filament with 14-nm periodicity. By combining the data from these electron microscope studies and the peptide mapping and competitive binding studies we have established the binding sites of 16 of 23 monoclonal antibodies. Two of the 23 antibodies block the formation of myosin-II filaments and given sufficient time, disassemble preformed myosin-II filaments. Both antibodies bind near one another at the tip of the myosin-II tail and are those that decorate the bare zone of preformed bipolar filaments with 14-nm periodicity. None of the other antibodies affect myosin filament formation, including one that binds to another site near the tip of the myosin-II tail. This demonstrates that antibodies can inhibit polymerization of myosin-II, but only when they bind to key sites on the tail of the molecule.     

Cryo-atomic force microscopy of smooth muscle myosin.

The motor and regulatory domains of the head and the 14-nm pitch of the alpha-helical coiled-coil of the tail of extended (6S) smooth-muscle myosin molecules were imaged with cryo atomic force microscopy at 80-85 K, and the effects of thiophosphorylation of the regulatory light chain were examined. The tail was 4 nm shorter in thiophosphorylated than in nonphosphorylated myosin. The first major bend was invariant, at approximately 51 nm from the head-tail junction (H-T), coincident with low probability in the paircoil score. The second major bend was 100 nm from the H-T junction in nonphosphorylated and closer to a skip residue than the bend (at 95 nm) in thiophosphorylated molecules. The shorter tail and distance between the two major bends induced by thiophosphorylation are interpreted to result from melting of the coiled-coil. An additional bend not previously reported occurred, with a lower frequency, approximately 24 nm from the H-T. The range of separation between the two heads was greater in thiophosphorylated molecules. Occasional high-resolution images showed slight unwinding of the coiled-coil of the base of the heads. We suggest that phosphorylation of MLC20 can affect the structure of extended, 6S myosin.     

Electron microscopic mapping of monoclonal antibodies on the tail region of Dictyostelium myosin

The binding sites of five monoclonal antibodies against myosin of Dictyostelium discoideum have been mapped. These antibodies bind to the tail region of the myosin molecule. By rotary shadowing, images of myosin-antibody complexes were obtained in which the mean distance of the midpoint of an antibody molecule from the myosin heads was localized with a precision better than 2 nm (90% confidence limit). Other quantitative data extracted from electron micrographs provided information on the stoichiometry of antibody-myosin interaction. Certain antibodies interacted with myosin molecules only at a ratio of 1:1. Other antibodies formed complexes of two molecules bound to homologous sites on a double-stranded myosin tail. Affinities were estimated and the abilities of different antibodies to cross-connect two myosin molecules were evaluated.