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 seg3^7,43 (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.