Conformational state of myosin's disordered loop 2 structure mediates actomyosin association during crossbridge formation.

The binding of myosin to actin to form crossbridges is a critical step for force generation by sarcomeres. A recent cryo-electron microscopy structure has resolved the weakly-bound actomyosin complex (AM.ADP.Pi). However, the structural and dynamic factors that influence actin-myosin association are unclear. The disordered loop 2 of myosin is thought to mediate actomyosin interactions in a complex, chemomechanical state-dependent fashion. Loop 2 is usually unresolved in structural studies due to its intrinsic disorder. Here, we utilize a combination of molecular dynamics simulations and electrostatic calculations to investigate the dynamics of these actin binding regions of myosin. Our results show that loop 2 experiences disordered dynamics and that specific conformations sampled modulate the strength of the associative electrostatic force between actin and myosin. Variation in the actin-myosin associative force was associated with the presentation and orientation of positively charged residues in loop 2. We provide an in-depth analysis of the conformational state space occupied by loop 2 during molecular dynamics simulations of pre-powerstroke human β-myosin S1, with three 500 ns replicates each of wildtype and two different mutant (E525K and V606M) myosin structures. This data set allowed for exploration of how loop 2 conformational sampling is altered by these two mutations which have experimentally been shown to alter actin binding affinity and clinically associated with cardiomyopathy. The E525K and V606M mutations altered the conformational ensemble sampled by loop 2 and were associated with associative actin binding strength. These results highlight the importance of the positive charges on loop 2 for actomyosin interactions and demonstrate how disease-causing mutations outside of loop 2 can still affect it.