Defining Myosin Relay Domain Interactions with the Converter Domain and with the SH1-SH2 Helix Region and their Significance in Muscle Contraction

Abstract

Interaction of the myosin relay domain with the converter domain and with the SH1-SH2 helix region appears to be vital for the power stroke and muscle contraction. To define involvement of specific residues in these interactions we used mutation and compensatory mutation approach. The mutant myosins were expressed transgenically in Drosophila indirect flight muscles, which allow assessment of myosin function in vitro and in vivo. We mutated residue R714 near the SH1-SH2 helix in the main body of myosin. We found that R714E myosin ATPase is reduced by ∼80% and that no in vitro motility is detected. R714E organisms show myofibril assembly defects that are exacerbated with age and these organisms cannot fly. Putative compensatory mutation E499R in the relay domain improves ATPase activity so that there is only ∼35% reduction compared to wild type. Further, R714E/E499R myosin moves actin filaments at ∼30% of wild-type velocity. Normal myofibril assembly is restored and myofibrils are more stable. We next studied mutation R759E in the converter domain. It depresses myosin ATPase activity, in vitro motility and flight ability. Although myofibrils assemble normally, they degenerate as flies age. We inserted a lysine mutation at putative interacting relay domain loop residue N509 in an attempt to suppress the R759E mutant phenotypes. N509K dramatically rescues muscle structure and function, with significant increases in ATPase rates and in vitro motility. These interactions are critical for normal ATPase activity, in vitro motility, myofibril assembly and/or stability and flight muscle function. Thus the specific residue contacts identified permit the myosin relay domain to communicate information between the nucleotide binding pocket and the converter domain and are essential for progress through the mechanochemical cycle that powers muscle contraction.