Structural Thermodynamics and Kinetics of the Cardiac Myosin/Omecamtiv Mecarbil Complex Revealed by Time-Resolved FRET

We are investigating the structural thermodynamics and kinetics of cardiac myosin in the presence of omecamtiv mecarbil (OM), a small molecule that is currently in clinical trials for treating heart failure. AM has been shown to increase thin-filament activated cardiac myosin ATPase activity, the apparent rate constant for actin-activated phosphate release from cardiac myosin, and contractility in the heart (Malik et al, Science 2011). How OM modulates theses activities remains poorly understood. Based on a published Biophysical Society Annual Meeting poster abstract (Liu et al. Biophys. J. 2013) and on published studies (Malik et al, Science 2011), we hypothesized that OM stabilizes the pre-powerstroke structural state in the presence of ATP analogs and increases the observed rate constant for the actin-activated powerstroke during actin-activated single ATP turnover. To test these predictions, we used a time-resolved FRET biosensor of β-cardiac myosin, which detects the mole faction of pre and post-powerstroke structural states of the myosin light-chain binding domain, to determine the apparent equilibrium constant for the recovery-stroke under saturating ADP, ADP.BeF, ADP.AlF and ADP.V nucleotide states in the presence of saturating OM or DMSO. We varied the temperatures of these experiments to determine whether OM changes the structural thermodynamics of the pre-powerstroke state. In a second set of experiments, we directly measure light-chain domain rotation during ATP binding to myosin and during actin binding to myosin in the presence of ATP. Taken together, these results address two of the key phenotypes that the drug exhibits in vitro, increased actin-activated phosphate release, and increased thin-filament activated steady-state ATPase cycling.