Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape1. The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients2,3. The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail3,4,5,6,7, creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited7,8. This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells9,10,11,12, which can be activated to form functional filaments as needed13—but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction.
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Structural data that support the findings of this study on the structure of 10S myosin II have been deposited in the Electron Microscopy Data Bank under accession code EMD-22145 (the electron microscopy density map) and in the Protein Data Bank under accession code 6XE9 (the atomic model). PDB data used to build the initial model were PDB 1I84 and PDB 2FXM.
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This work was supported by National Institutes of Health grants AR072036, AR067279, and HL139883 (R.C.), HL075030, HL111696 and HL142853 (M.I.), and a University of Texas STARs PLUS Award (M.I.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank K. Subramanian, C. Ouch, W. Royer, C. Xu, C. Gaubitz and N. Stone for advice and discussion on fitting and refinement, K. K. Song and C. Xu for cryo-EM imaging, L. Alamo and A. Pinto for advice on homology modelling, and M. Espinoza-Fonseca for providing the atomic models of the RLC NTEs. Cryo-EM imaging was carried out in the Massachusetts Facility for High-Resolution Electron Cryo-Microscopy at the University of Massachusetts Medical School. The Titan Krios was purchased with a grant from the Massachusetts Life Sciences Center capital fund. UCSF Chimera, used for molecular graphics and analyses performed in this work, is supported by National Institutes of Health grant GM103311.
The authors declare no competing interests.
Peer review information Nature thanks Gregory M. Alushin, Robert Cross and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Raw cryo-EM image. Circles indicate individual molecules; red, face view; blue, edge view. This is one of 10,950 micrographs recorded at 300 kV on the Titan Krios, and is representative of those showing the particles most clearly. A preliminary set of 400 micrographs from a different set of grids was first recorded on a Talos Arctica at 200 kV, producing a similar, initial reconstruction, at 9 Å resolution. b, Gold-standard Fourier shell correlation (FSC) curve using half maps; global resolution estimate is 4.3 Å by the FSC 0.143 criterion. c, Typical 2D class averages of 10S myosin (25 of a total of 43 good class averages, representing a total of 260,360 particles). Edge views show poor definition of the longer end of the tail (stars), corresponding to mobility in the upper part of the reconstruction (Extended Data Fig. 2). d, Enlarged class average showing main features in reconstruction. BH/FH = blocked/free head; Seg = segment number.
a, b, Front and rear views of density map (contour level 0.0125), showing estimated local resolution according to RESMAP51. Resolution of the heads is highest in the MDs (especially the BH), and lower in the RDs (especially the FH), corresponding to local regions of varied mobility. Resolution in the tail is best where it is stabilized by contacts with other domains (seg2 with the BH MD and ELC), especially in the specific α-helix making the contact. The tail regions at the top are noisy and of low density (Extended Data Fig. 1c). c, d, Docking of the refined model to the map shown at high contour cutoff (0.025; cf. Fig. 2a), revealing clear secondary structure and quality of fit (front, rear views respectively). e–g, Fitting to show map quality. e, β-strand 249-254 of the BH MD, showing side-chain density. f, α-helix 431-443 in the BH MD, showing side-chain density. g, Coiled-coil in seg2 contacting the BH MD (1425-1491), showing 5.4 Å α-helical pitch (cf. d).
a, Superposition of BH (red) and FH MDs (green) using Matchmaker in Chimera. There is an almost perfect match, including the converter domains (Cnv), which show no more than ~5 Å movement, suggesting that the two heads are in the same biochemical state. Orientation of heads is that seen in front view of BH. b, c, Alignment of MDs of BH and FH, oriented as attached to actin in rigor state. There is a large difference in angle between the BH and FH RDs with respect to their MDs in both longitudinal (b) and azimuthal planes (c) (defined with respect to plane of filament sliding)14. BH and FH were aligned by superposing their MDs on the MD (not shown) of mammalian actomyosin in the rigor state (PDB 5H53). RLCs and ELCs have been removed for clarity. d, Face-view of IHM shows how C termini of the two heads (P849), at bottom of RDs, come within ~28 Å of each other where they meet S2 (not shown). This proximity depends on the differential flexing of the RDs with respect to the MDs in the two heads. If the FH had the BH RD angle, the C termini in the IHM would be too far apart (~ 58 Å) to join to the 20 Å-diameter S2 without its substantial uncoiling. The angle of the FH RD is the major structural difference that brings the C termini of the two heads close enough together to make their simultaneous attachment to S2 possible. Comparison of RD angles was made by superposing the BH and FH MDs. e, Comparison of isolated regulatory domain structures in BH and FH (LCs omitted for clarity) after aligning residues K823-P849 (the left half of the molecule). The C-terminal hooks make similar ~90° angles with the RD helix. There is a small difference in angle between the FH and BH helices in the N-terminal half of the RD heavy chain. f, Comparison of regulatory domain structures within IHM. The BH RD was superimposed on the FH RD in the N-terminal half. The straighter course of the FH RD brings the FH and BH C termini that attach to S2 closer together by ~7 Å, facilitating attachment to S2 without any substantial unwinding of the coiled coil. This flexibility in the RD, bringing the FH C terminus closer to the BH, thus aids in formation of the IHM, along with the different angles of the RDs with respect to the MDs seen in b–d.
a, Atomic model fitted into the electron density map (contour level 0.021) in the region of the head-tail junction. The map shows tubes of density for the two hooks, which form 90° bends with the α-helical heavy chain of the RDs. Such tubular density is characteristic of α-helices in other parts of the structure. P849 is the invariant proline that marks the junction between each head and the tail. View is from front. b, IHM front-view showing hooks at C-terminal end of each RD. ELCs and RLCs removed for clarity. c, Enlargement of hook region showing ~28 Å distance between invariant prolines at the C terminus of each head heavy chain. d, IHM face-view showing α-helical backbone of BH and FH RDs, hooks, and coiled-coil tail regions. Seg1 α-helices (red, green) continue from BH and FH hooks.
a, Oblique view of back of reconstruction. Red arrows indicate relatively regular crossovers of coiled-coil (~60-75 Å apart) as segment 2 wraps around the BH MD, and a long, parallel (untwisted) stretch in segment 3 after leaving hinge 2 (~100 Å; white bar). b, Atomic model of tail showing approximate distance between crossovers in coiled coil. Purple residues (numbered) are estimated to be at crossovers as seen in this face view, with distances between them shown. E1535-E1612 is an almost straight, non-coiled region of the tail, especially the green chain. The two heads would connect to the bottom of seg1 at L850. L850-T889 represents the first ~ 5 heptads of S2 (black star), which appear to associate with each other from the start, though with a slightly longer helical pitch in the first few residues. The two α-helices are shown in different colours for clarity. Their specific connection to the BH or FH is unknown (apart from seg1) due to lack of continuity of density in the top half of the reconstruction. Blue region in seg2 is M1462-K1472 (see text). c, Side-view of reconstruction showing the long stretch of untwisted coiled-coil in segment 3, running over the BH. Yellow star here and in a shows position of skip residue 1592. d, Hinge 2, in face- and end-views, showing continuity of coiling of the two α-helices about each other through this sharp bend, with local melting of α-helices likely at the bend. Glu1535 (spheres) is thought to mark the hinge point, although uncertainty in measurement of negative stain images means that the hinge could occur 2-3 amino acids either side of 15354. For this reason, numbering of amino acids in segs 2 and 3 in the atomic model is uncertain to the same degree. Density maps shown at contour level of 0.016.
The different position of seg1 in the IHM of the tarantula filament and the 10S molecule is illustrated by comparing the PDBs of the best fits (tarantula filament, PDB 3JBH; 10S molecule, PDB 6XE9 [this work]). a, b, Front and rear views of IHM in which the head regions (filament, tan; 10S, blue) have been superposed (using Matchmaker in Chimera). c, d, Same views as for a, b, but with heads removed for clarity. Seg1 is yellow for the filament (bent conformation) and pink for the molecule (straight). The two segments run in different positions, ~ 20 Å apart, centre to centre. Strikingly, the position occupied by seg1 in the filament is taken by seg3 in the 10S molecule (yellow/blue overlap). e, f, The different seg1 positions are also clear when the filament (red) and 10S (blue) maps are compared. Front and rear views confirm that seg1’s in the two reconstructions are laterally displaced from each other. Similar results are obtained for two independent filament maps: EMD-1950 (shown)36 and EMD-651260.
a–g, Converter domain inhibition. a, Atomic model of 10S structure. Both heads are in the ADP.Pi state (see text). b, 10S model, with a myosin head in ADP state (PDB 3I5F, yellow heavy chain) superimposed on the BH by matching motor domains. The RD in this nucleotide state is straight and the Cnv (pink) is in a very different location from the BH Cnv (purple), clearly clashing with seg2. c, d, Detail of this clash in front and end views, with other parts of molecule removed. This comparison shows that for the BH to lose its Pi (going to the ADP state), its Cnv will clash with seg2. We conclude that seg2 acts as a physical barrier (reinforced by the mechanical restraint created by the connection of both lever arms to S2), preventing this transition and inhibiting BH ATP turnover. e, 10S model, with ADP-state head (PDB 3I5F) superposed on the FH MD. f, g, Detail of e, with LCs removed for clarity. The ADP-state RD and its Cnv (pink) are to the right (red arrow) of the ADP.Pi-state FH. This comparison suggests that for the FH to lose its Pi, the interaction of its Cnv with the BH MD must be broken. We propose that this is inhibited in the 10S structure by the strength of this interaction (BF), reinforced by FH interactions with the tail (TF1, TF2 and TF3). Together these interactions would prevent the ADP.Pi→ADP transition and inhibit FH ATP turnover. h–k, Actin-binding inhibition. The BH and FH were attached to actin by superposing their MDs on the MD (not shown) of mammalian actomyosin in the rigor state (PDB 5H53), as described in Extended Data Fig. 3. Segments 2 and 3 were removed for clarity. The modelling shows that attachment via both BH and FH is inhibited due to major steric clashes of other parts of the 10S structure with actin. h, Inhibition of binding via the FH. Front view of IHM shows that S2 clashes (dashed circle) with actin (2 monomers shown). i, Rotated 90° around vertical axis with respect to h. j, Inhibition of binding via the BH. Front view of IHM shows that S2 and the FH both clash with actin (dashed circles). k, Rotated 90° around vertical axis with respect to j.
Extended Data Fig. 8 Proposed mechanism of 10S myosin inhibition and activation, based on the atomic model and MD simulations.
Smooth and nonmuscle myosin IIs are activated by phosphorylation of their RLCs on S19, leading to breaking of the 10S intramolecular interactions, unfolding to the extended structure, and assembly into functional filaments. Our atomic model indicates a possible mechanism. Our previous work suggested that the single interaction most critical to the folded conformation is that occurring between seg3 and the BH RD7, and we noted how proximity of BH RLC S19 might regulate this interaction. Our atomic model suggests that seg3 in fact contacts the BH RLC at two sites. One is the C-lobe (TB5, Fig. 3a, g, and panel e above). The other involves the 24-residue N-terminal extension of the RLC, the phosphorylation domain (PD61), containing S19 (interaction TB6, Fig. 3a). The PD is not observed in structures of the myosin head, but has been modelled by molecular dynamics simulations (a(i), dephosphorylated PD, ribbon and surface charge depictions; red negative, blue positive; upper box, PD sequence, MLCK binding site green; S19, yellow; N-terminal half positively charged)61. Our EM map reveals significant density (b, red rectangle), extending from F25, that fits this PD (b shows best fit of model from a(i) to BH PD density) and lies over seg3, below TB5 (red rectangles in b, showing fitting; c, model based on fit; e, zoomed-out model). In the atomic model (c), interaction occurs between positively charged residues of the PD N-terminal half and a negatively charged patch (~1560-1572) in seg37,43 (d, red rectangle; surface charge depiction; red, negatively charged; blue, positively charged; see interaction TB6 in Supplementary Table 1), which could strengthen TB5 (e). There is also significant density for a portion of the FH PD (b, green rectangle), which fits residues 20-24, while the positively charged N-terminal half (a) fits weak density near to negatively charged residues of BH RLC helix B (b-e, green rectangles). This would strengthen interaction BF2 between the RLCs. These interactions involving the RLCs, especially the BH PD with seg3, appear to be the key features creating the off state, supported by the other interactions already described. The structural basis of unfolding upon S19 phosphorylation remains unknown due to the absence of the PD in previous structures. The apparent PD densities we observe suggest the following model (h). Phosphorylation appears to occur first on the FH, then the BH7. EPR and molecular dynamics simulations suggest that phosphorylation causes straightening and stiffening of the PD61 (a: i. dephosphorylated, ii. phosphorylated, iii. transition, dephosphorylated → phosphorylated). When the unphosphorylated PDs (compact in our map; e, h, stage 1 in the activation sequence) are replaced by the phosphorylated (straightened) conformations (grey helices in f, g, using PD structures from a(ii)), the FH PD interaction with BH RLC helix B is removed (due to straightening and to the reduction in positive charge), which could weaken the RLC-RLC and thus head-head interaction (f, FH RLC phosphorylated, purple rectangle)36, releasing the FH, while retaining the folded tail structure (h, stage 2). When the BH is also phosphorylated, straightening/stiffening of its PD, and reduction in its positive charge, breaks its interaction with seg3 (g, red arrow, yellow rectangle; h, stage 3). With weakening of these interactions, seg2 could dissociate from the BH MD and ELC, leading to complete unfolding to the 6S structure (h, stage 4). In support of this proposal, replacement of charged amino acids near S19 in the RLC PD showed that unfolding upon phosphorylation may be due to net charge reduction of the PDs62. This physical model suggests that the two PDs with their phosphorylation sites, and the associated regions of seg3, represent a localized structural confluence in which the key events of activation and deactivation take place (the “phosphorylation zone”, e-g). We tested the PD structure suggested by the MD simulations (in the case of the BH) by examining the sharpened map in this region and manually creating a model with the PD sequence to best fit the map using Coot (panel (i) above; viewing angle changed slightly from b to best show density and model features). The density clearly suggests a short helix followed by a loop and a second helix, with density present for the entire length of the PD. This is the first time that the PD has been directly visualized, as it is disordered in isolated myosin heads. We suggest that it is the binding of the PD to seg3 (occurring only in the 10S structure) that makes this visualization possible. The atomic model based on this fitting broadly supports the bent, helix–loop–helix conformation suggested by the MD simulations of the unphosphorylated PD (a(i)). The model (panel i) suggests that basic residues K11, K12, and R13, close to acidic residues D1565 and E1566 in seg3, electrostatically hold seg3 in the folded conformation—the most crucial interaction of the 10S structure—and in close proximity to the regulatory S19. MD simulations suggest that phosphorylation creates a salt bridge between phosphorylated S19 and R16, which causes the PD loop to become α-helical, straightening and stiffening the PD as a whole61. As discussed above, we propose that it is this straightening, and the reduction in positive charge of the PD, that cause the dissociation of seg3 from the PD, leading to unfolding and activation of the 10S structure as a whole (a, g, h). From the model it is not clear whether the BH RLC would be fully available for binding by MLCK in the 10S structure. Importantly, even if sufficiently exposed, the interaction of K11-R13 with seg3 could slow binding by MLCK, as these residues are also involved in MLCK substrate recognition63. If such hindrance occurs, this would be consistent with the proposal7 that BH phosphorylation, occurring after FH phosphorylation (h), is the final, required step for activation and unfolding.
a, Overview of 10S molecule showing distribution of mutations, yellow in tail, blue in heads. b–d. Enlargements of mutation regions. b, Proximity of BH converter (Cnv, purple) and SH3 domain (pink) to seg2 mutations, and of BH SH3 and MD mutations to seg2. c, Mutations in segs 1, 2, and 3. V1529 is near hinge 2 and could impact hinge function. R1570 is part of the proposed interaction region of the BH RLC PD on seg3, and could impact RLC function. M860-E866 is a duplication, which could translate seg1, impacting its interactions downstream. d, Mutations in seg1 coincident with TT2 interaction. See Fig. 2b, Extended Data Table 2.
10S myosin intramolecular interactions. The Table lists the intramolecular interactions at each site of contact in the 10S structure, with a labelled image of each. The table correlates with Fig. 3 of the text.
Sequence alignment of myosin II heavy chains. The Table shows the alignment of the heavy chain sequences of nonmuscle myosin IIA, B, and C and smooth muscle myosin. This alignment is used in plotting mutations in the different myosins onto the 10S structure (Fig. 2b, c; Extended Data Fig. 9, Extended Data Table 2).
3D map of 10S myosin. The 10S myosin map (EMD-22145) is rotated about Y- and X-axes. BH: heavy chain (HC), red; ELC, dark blue; RLC, orange. FH: HC, green; ELC, pale blue; RLC, yellow. SH3 domains, pink; converter domains, purple. Seg1, forest blue; seg2, cyan; seg3, magenta. See Fig. 1.
3D map of 10S myosin fitted with refined atomic model. The translucent map (EMD-22145) fitted with refined model (PDB 6XE9) is rotated about Y- and X-axes. BH: HC, red; ELC, dark blue; RLC, orange. FH: HC, green; ELC, pale blue; RLC, yellow. SH3 domains, pink; converter domains, purple. Seg1, forest blue; seg2, cyan; seg3, magenta. See Fig. 2a.
Refined atomic model of 10S myosin (6XE9). The model is rotated about Y- and X-axes. BH: HC, red; ELC, dark blue; RLC, orange. FH: HC, green; ELC, pale blue; RLC, yellow. SH3 domains, pink; converter domains, purple. Seg1, forest blue; seg2, cyan; seg3, magenta.
Refined atomic model of 10S myosin showing mutation sites. The model (PDB 6XE9) is rotated about Y- and X-axes, showing mutation sites, coloured dark blue in heads and yellow in tail. BH: HC, red; ELC, dark blue; RLC, orange. FH: HC, green; ELC, pale blue; RLC, yellow. SH3 domains, pink; converter domains, purple. Seg1, forest blue; seg2, cyan; seg3, magenta. See Fig. 2b, Extended Data Table 2.
Refined atomic model of 10S myosin showing putative interaction sites. The model (PDB 6XE9) is rotated about Y- and X-axes, showing intramolecular interaction sites, coloured cyan and yellow to distinguish the interacting partners. BH: HC, red; ELC, dark blue; RLC, orange. FH: HC, green; ELC, pale blue; RLC, yellow. SH3 domains, pink; converter domains, purple. Seg1, forest blue; seg2, cyan; seg3, magenta. See Fig. 3, Supplementary Table 1.
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Yang, S., Tiwari, P., Lee, K.H. et al. Cryo-EM structure of the inhibited (10S) form of myosin II. Nature 588, 521–525 (2020). https://doi.org/10.1038/s41586-020-3007-0
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