Abstract
An understanding of the conformational changes underlying activation of regulated myosins will offer important insights into the mechanism and regulation of contraction. Certain sequence features of vertebrate smooth myosin, however, have interfered with efforts to crystallize key portions of the molecule and to obtain atomic resolution structures. Vertebrate smooth myosin is activated by calcium-triggered phosphorylation of its regulatory light chain (RLC), whereas most invertebrate myosins are activated by direct binding of Ca2 ·'· to the essential light chain (ELC). The "catch" muscle of the sea scallop P/acopecten mage/lanicus, however, contains two RLCs, one of which (SmoA) has the phosphorylation sequence of vertebrate smooth myosin RLC. Moreover, the head (or subfragmcnt-1, Sl) of Placopecten catch muscle containing both light chains has already been crystallized (albeit with a cleaved RLC) (Yang et al., 2007). This finding suggests that the RD of this sea scallop catch muscle myosin, including its unique RLC, may crystallize far more readily than that of the vertebrate smooth myosin. The SmoA RLC (from a previous eDNA expression study; Perreault-Micale eta!., 1996a) was used to reconstitute a regulatory domain (RD) which is capable of mediating myosinregulated contraction both by phosphorylation and by Ca2+ -binding. The ELC and the heavy chain fragment (HCF, the portion of Sl that is included in the RD) were isolated from sea scallop striated muscle, because their amino acid sequence~ are identical to their counterparts in the scallop catch muscle (Perreault-Micale eta!., 1996b). We aimed to crystallize this RD under four conditions: neither Ca2+-bound nor phosphorylated, Ca2+-bound only, phosphorylated only, and both Ca2+ -bound and phosphorylated. This RD has now been crystallized and X-ray data sets have been collected for protein prepared under both of the Ca2+-containing conditions. The structure has thus far been determined for the unphosphorylated Ca2+-bound RD. When the atomic structures of the conformations assumed under each of these four conditions are solved, key intbm1ation about the contractile mechanism and possibly the regulation of diverse regulated myosins may' be inferred.