Actin Straightens the Myosin Relay Helix during the Powerstroke

Abstract

We have used transient time-resolved FRET, (TR)2FRET, to resolve the structural kinetics of the myosin relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in all myosin isoforms coupling structural changes in the ATPase site with rotation of the light chain binding domain and force generation. Previous work demonstrated that the relay helix of the myosin II motor domain from Dictyostelium Discoideum exists in dynamic equilibrium between a bent pre-powerstroke state stabilized by ATP, and a straight post-powerstroke state which predominates in the presence of ADP or when the nucleotide binding site is empty. We predict that actin binding to the myosin.ADP.Pi complex will reverse helix bending. This transition should parallel the myosin power-stroke. To test this hypothesis, a Cys-lite Dictyostelium myosin motor domain was labeled at two introduced Cys residues shown to detect relay helix bending using DEER, TR-FRET and (TR)2FRET spectroscopy. We preformed a (TR)2FRET experiment measuring the structural distribution of the relay helix every millisecond after rapid mixing of myosin.ADP/Pi with actin. This experiment showed that actin binding straightens the bent helix. We characterized the actin dependence of helix straightening and then correlated this dependence with the kinetics of actin binding and phosphate dissociation from the myosin active site. Numerical simulations were used to model a system containing pre and post-powerstroke structural states, actin binding, hydrolysis and phosphate dissociation. We globally fit this model to the (TR)2FRET, actin binding, and phosphate dissociation transients. From this analysis, we are able to determine the timing of helix straightening with respect to actin binding and the critical step of phosphate dissociation which acts as the thermodynamic driving force for myosin's force generating powerstroke.