Contraction of muscle involves the cyclic interaction of myosin heads on the thick filaments with actin subunits in the thin filaments. Muscles relax when this interaction is blocked by molecular switches on either or both filaments. Insight into the relaxed (switched OFF) structure of myosin has come from electron microscopic studies of smooth muscle myosin molecules, which are regulated by phosphorylation. These studies suggest that the OFF state is achieved by an asymmetric, intramolecular interaction between the actin-binding region of one head and the converter region of the other, switching both heads off. Although this is a plausible model for relaxation based on isolated myosin molecules, it does not reveal whether this structure is present in native myosin filaments. Here we analyse the structure of a phosphorylation-regulated striated muscle thick filament using cryo-electron microscopy. Three-dimensional reconstruction and atomic fitting studies suggest that the 'interacting-head' structure is also present in the filament, and that it may underlie the relaxed state of thick filaments in both smooth and myosin-regulated striated muscles over a wide range of species.
SummaryMuscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the freehead and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens reveals that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal here has been to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction reveals intra-and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC, and fitted to the reconstruction an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 crystal structure. The fitting suggests an intramolecular interaction between the cardiomyopathy loop of the free-head and its own S2 and two intermolecular interactions-between the cardio-loop of the free head and the ELC of the blocked head, and between the Leu-305 -Gln-327 "interaction loop" (loop I) of the free-head and the N-terminal fragment of the RLC of the blocked-head. These interactions, added to those previously described, would help to switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.
Regulation of muscle contraction via the myosin filaments occurs in vertebrate smooth and many invertebrate striated muscles. Studies of unphosphorylated vertebrate smooth muscle myosin suggest that activity is switched off through an intramolecular interaction between the actin-binding region of one head and the converter and essential light chains of the other, inhibiting ATPase activity and actin interaction. The same interaction (and additional interaction with the tail) is seen in three-dimensional reconstructions of relaxed, native myosin filaments from tarantula striated muscle, suggesting that such interactions are likely to underlie the off-state of myosin across a wide spectrum of the animal kingdom. We have tested this hypothesis by carrying out cryo-electron microscopy and 3D image reconstruction of myosin filaments from horseshoe crab (Limulus) muscle. The same head-head and head-tail interactions seen in tarantula are also seen in Limulus, supporting the hypothesis. Other data suggest that this motif may underlie the relaxed state of myosin II in all species (including myosin II in nonmuscle cells), with the possible exception of insect flight muscle. The molecular organization of the myosin tails in the backbone of muscle thick filaments is unknown, and may differ between species. X-ray diffraction data support a general model for crustaceans in which tails associate together to form 4 nm diameter subfilaments, with these subfilaments assembling together to form the backbone. This model is supported by direct observation of 4 nm diameter, elongated strands in the tarantula reconstruction, suggesting that it might be a general structure across the arthropods. We observe a similar backbone organization in the Limulus reconstruction, supporting the general existence of such subfilaments.
Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca 2+ binding to the essential light chains, contributing to on-off switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the two myosin heads, inhibiting their actin binding or ATPase activity. Here, we have tested whether a similar interaction switches off activity in myosin filaments regulated by Ca 2+ binding. Cryo-electron microscopy and single-particle image reconstruction of Ca 2+ -regulated (scallop) filaments reveals a helical array of myosin headpair motifs above the filament surface. Docking of atomic models of scallop myosin head domains into the motifs reveals that the heads interact in a similar way to those in phosphorylation-regulated filaments. The results imply that the two major evolutionary branches of myosin regulation-involving phosphorylation or Ca 2+ binding-share a common structural mechanism for switching off thick-filament activity in relaxed muscle. We suggest that the Ca 2+ -binding mechanism evolved from the more ancient phosphorylation-based system to enable rapid response of myosin-regulated muscles to activation. Although the motifs are similar in both systems, the scallop structure is more tilted and higher above the filament backbone, leading to different intermolecular interactions. The reconstruction reveals how the myosin tail emerges from the motif, connecting the heads to the filament backbone, and shows that the backbone is built from supramolecular assemblies of myosin tails. The reconstruction provides a native structural context for understanding past biochemical and biophysical studies of this model Ca 2+ -regulated myosin.scallop muscle | molluscan muscle | thick-filament structure | 3D reconstruction | muscle regulation C ontractile activity in muscle is switched on and off by protein subunits on the thick (myosin-containing) and thin (actincontaining) filaments (1). Regulation via myosin is based on either Ca 2+ -dependent phosphorylation of the myosin regulatory light chains (RLCs) (this mode of regulation occurs in vertebrate smooth muscle and some invertebrate striated muscles) or Ca 2+ binding to the essential light chains (ELCs) (occurring in some invertebrate striated muscles) (2-6). In some muscles (vertebrate striated and some invertebrate striated), phosphorylation modulates activity but is not required for muscle activation (2,7,8). Electron-microscopic studies of vertebrate smooth muscle myosin molecules have revealed that, in the relaxed (dephosphorylated) state, the two myosin heads in a molecule interact with each other asymmetrically so that the actin-binding region of one (the "blocked" head) is blocked by interaction with the converter domain and ELC of the other (the "free" head). It is thought that this switches off actin-binding and ATPase activity of the blocked and free heads, respectively (9, 10), contributing to muscle relaxation. Phosphorylati...
Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100 microM blebbistatin, disordering was at least 10 times slower. In the M.ADP state, myosin heads are also disordered. When blebbistatin was added to M.ADP thick filaments, helical ordering was restored. However, blebbistatin did not improve the order of thick filaments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and filament sliding. In the presence of blebbistatin, filament interaction was inhibited. These structural observations support the conclusion, based on biochemical studies, that blebbistatin inhibits myosin ATPase and actin interaction by stabilizing the closed switch 2 structure of the myosin head. These properties make blebbistatin a useful tool in structural and functional studies of cell motility and muscle contraction.
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