Modulation of myosin mechanics and kinetics by the myosin regulatory light chain

The myosin regulatory light chain (RLC) is a calmodulin homology protein that binds to the alpha-helical myosin neck region. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate force and motion. The role of the RLC as a structural element is well established; however, its role in modulating the kinetics and mechanics of actomyosin contractility is not as well understood. In order to explore the role of the RLC in modulating actomyosin contractility in muscle, we examined how modifications to the N-terminus of the RLC affect myosin kinetics and mechanics at the molecular level using an array of in vitro motility assays. In particular, we examined the effects of RLC phosphorylation in skeletal muscle and the effects of cationic binding site mutations of cardiac muscle RLC that have been implicated in familial hypertrophic cardiomyopathy (N47K and R58Q). Our data show that although the RLC is spatially separated from the myosin active site, it appears to have a significant role in modulating contractility and that small changes to the RLC can cause significant alterations in the kinetics and mechanics of actomyosin based motility. The exact changes in myosin kinetics and mechanics induced by modification of the RLC depend on both position of the alteration along the RLC as well as the isoform of the myosin heavy chain to which the RLC binds. Furthermore, we show that subtle changes to the RLC can profoundly affect both the unloaded and loaded myosin kinetics, suggesting a role for the RLC in modulating strain dependent mechanochemistry. Specifically, whereas phosphorylation of the myosin RLC appears to increase strain sensitivity, the RLC mutations implicated in familial hypertrophic cardiomyopathy appear to decrease strain sensitivity. We propose that these changes in strain sensitivity are physiologically relevant and that changes in strain sensitivity in the myosins bearing mutant RLCs may lead to the observed disease phenotypes.