John A. Rohde

Direct real-time detection of the structural and biochemical events in the myosin power stroke

Significance Myosins use ATP hydrolysis to power movement, but how they do this remains enigmatic. If we understood exactly how ATP’s chemical energy is coupled to force generation, we would gain insight into the fundamental biophysics of these fascinating and medically important proteins. We combined subnanosecond time-resolved FRET with millisecond-resolved transient kinetics to monitor subnanometer structural transitions in myosin during the actin-activated myosin power stroke. Our results show that the power stroke precedes phosphate release, thus contradicting the dominant model of myosin function, which argues that phosphate release should precede the power stroke. Our work prompts future studies investigating how this coordination is altered by disease-causing mutations and therapies designed to treat these diseases. A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)2FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin’s power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.

Heart failure drug changes the mechanoenzymology of the cardiac myosin powerstroke

Significance Heart failure is the leading cause of mortality in the United States, despite sustained efforts to develop effective small-molecule treatments. The biophysical characterization of existing therapies will drive development of next-generation approaches for treating heart failure. Furthermore, small molecules can be powerful probes for dissecting protein structure function relationships. We used an innovative FRET-based spectroscopic approach to determine that the small-molecule heart disease therapeutic omecamtiv mecarbil (OM) changes how myosin’s working powerstroke is coordinated with actin-activated phosphate release, the biochemical step associated with force generation. This result explains how OM alters cardiac contractility at the molecular level, forcing the accumulation of actin-bound prepowerstroke cross-bridges. Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin’s prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin’s ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin’s mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.

Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin

Significance Small-molecule allosteric effectors designed to target and modulate striated and smooth myosin isoforms for the treatment of disease show promise in preclinical and clinical trials. Beta-cardiac myosin is an especially important target, as heart disease remains a primary cause of death in the United States. One prevalent type of heart disease is hypertrophic cardiomyopathy (HCM), which is hypothesized to result from dysregulated force generation by cardiac myosin. Mavacamten is a potent cardiac myosin ATPase activity inhibitor that improves cardiac output in HCM animal models. Our results show that mavacamten selectively stabilizes a two-headed–dependent, autoinhibited state of cardiac myosin in solution. The kinetics and energetics of this state are consistent with the autoinhibited superrelaxed state previously observed only in intact sarcomeres. We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.