Structural Coordination of the Myosin Powerstroke

Abstract

Myosins drive actin-based motility required for important cellular functions and are model systems for enzyme based mechanochemical coupling. We are investigating the structural dynamics of myosins to determine how the movements of key structural elements are coordinated with the actin-activated ATPase cycling. We used FRET to investigate how the myosin powerstroke coordinates with actin-induced phosphate release in skeletal and cardiac myosins, two important myosin isoforms with disease relevance. In skeletal myosin, we find that weak actin binding triggers the myosin powerstroke before hydrolyzed phosphate dissociates from the myosin catalytic domain. We also find that the free energy change for the actin-induced light-chain domain rotation is small before phosphate is released, but large after it is released. This is consistent with a thermal diffusion model where the initiation of the myosin powerstroke is constrained by rapid diffusion of the light-chain domain between pre and post powerstroke orientations. In cardiac myosin, the kinetics of light-chain domain rotation and phosphate release are slower and not clearly resolved from each other. Interestingly, we find that addition of the cardiac myosin modulator omecamtiv mecarbil, accelerates actin-induced phosphate release, but slows the powerstroke. These results suggest that coordination of phosphate release and the myosin powerstroke is flexible, and that changing this coordination will change the force generating properties of myosin isoforms. Supported by: Graduate Excellence Fellowship-University of MN (JAR), NIH AR032961 & AR057220 (DDT), the Paul and Sheila Wellstone Muscular Dystrophy Center and American Heart Association (JMM).