Striated muscle contraction in most animals is regulated at least in part by the troponin-tropomyosin (Tn-Tm) switch on the thin (actin-containing) filaments. The only group that has been suggested to lack actin-linked regulation is the mollusks, where contraction is regulated through the myosin heads on the thick filaments. However, molluscan gene sequence data suggest the presence of troponin (Tn) components, consistent with actin-linked regulation, and some biochemical and immunological data also support this idea. The presence of actin-linked (in addition to myosin-linked) regulation in mollusks would simplify our general picture of muscle regulation by extending actin-linked regulation to this phylum as well. We have investigated this question structurally by determining the effect of Ca(2+) on the position of Tm in native thin filaments from scallop striated adductor muscle. Three-dimensional reconstructions of negatively stained filaments were determined by electron microscopy and single-particle image analysis. At low Ca(2+), Tm appeared to occupy the "blocking" position, on the outer domain of actin, identified in earlier studies of regulated thin filaments in the low-Ca(2+) state. In this position, Tm would sterically block myosin binding, switching off filament activity. At high Ca(2+), Tm appeared to move toward a position on the inner domain, similar to that induced by Ca(2+) in regulated thin filaments. This Ca(2+)-induced movement of Tm is consistent with the hypothesis that scallop thin filaments are Ca(2+) regulated.

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 head-pair 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.