Impact of salt concentration and free magnesium on human beta-cardiac myosin reveal important details about the conserved mechanochemical mechanism

The myosin ATPase cycle is dramatically accelerated by the presence of actin, while the structural details of actin-activation are still unclear. We found that a dilated cardiomyopathy mutation, E525K, in human beta-cardiac myosin subfragment 1 (S1) enhanced actin-activation of phosphate release and lever arm rotation ∼3-fold. We hypothesized that abolishment of the conserved 484 to 525 salt bridge allows for lysine 525 to more readily interact with actin. Indeed, we found that actin-activated ATPase activity of E525K was quite resistant to changes in salt concentration (20 mM to 100 mM KCl) compared to the WT motor, while in vitro actin-gliding velocities were similarly altered. Direct measurements of pyrene actin binding revealed that the E525K mutation accelerates attachment to actin. We found that the actin-activated ATPase activity of E525K was less sensitive to magnesium (Mg) than WT, indicating that the mutation allosterically alters the coordination of Mg in the active site. In addition, the Mg dependence of ADP release was significantly faster in the mutant at low Mg. Molecular dynamics simulations demonstrate that E525K increases the conformational variation of the active site in the ADP-bound state. Our results suggest that the E525K mutation enhances actin affinity by accelerating the transition from weak to strong actin binding, which highlights the importance of the activation loop/relay helix communication pathway during actin-activated Pi-release. In addition, E525K alters Mg coordination in the active site further demonstrating the importance of the 484 to 525 salt bridge in allosterically coupling the active site and actin-binding region.