Structural studies on the myosin molecular motor: A. Evidence for a 'detached' ATP site, and B. Comparison of calcium-bound and calcium-free regulatory domains

The molecular motor that drives muscle contraction is a protein known as myosin. Muscle contraction occurs when myosin heads bind to actin thin filaments and undergo a conformational change powered by ATP hydrolysis, pushing the actin filaments past the myosin thick filaments in a kind of rowing action known as a power stroke. The myosin head, which can be isolated as an active fragment known as Subfragment One (S1), is the site of both the enzymatic and motor activity. A recent structure of scallop myosin S1 complexed with MgADP featured the unwinding of the so-called SH1 helix, which, according to biochemical studies with cross-linkers such as p-PDM, only occurs in a weak actin-binding state, not an actin-bound S1-ADP state. Based on this and other biochemical evidence, the controversial suggestion was made that the structure actually mimics a detached ATP state, although ADP occupies the active site. To address this ambiguity, three new crystal structures are presented here of scallop S1 complexed with ATP analogs and/or p-PDM. In addition to demonstrating the same conformation as that of the previous S1-MgADP structure, two of the current structures reveal a surprise by the site of cross-linking by p-PDM.; Both the motor and enzymatic activities of scallop myosin are regulated by direct binding of Ca2+ to its regulatory domain (RD). In vertebrate smooth muscle, calcium-mediated phosphorylation of the RD controls myosin activity by a related mode of action. In an attempt to understand the “Off” state (no Ca2+ binding or phosphorylation) of regulated myosins, crystal structures have been determined for two Ca 2+-free scallop RD mutants, as well as a reconstituted (i.e. with two complete light chains) Ca2+-RD wildtype as a control. Although the mutant structures show no gross conformational changes from the Ca 2+-RD, predictions are made about the nature of the smooth muscle phosphate regulatory binding site and about possible interactions between myosin heads.