Myosins are essential molecular motors that drive muscle contraction and regulate cardiac output. A key mechanism for tuning myosin activity is the formation of the interacting heads motif (IHM), an autoinhibited conformation that sequesters myosin heads and reduces ATPase activity. Dysregulation of IHM stability is implicated in cardiomyopathies, yet the molecular mechanisms governing transitions out of the IHM state remain poorly understood due to the size and complexity of the system. We used long-timescale (>100 microseconds net sampling) molecular dynamics simulations to model dissociation of protein-protein interfaces that stabilize the cardiac β-myosin IHM. We performed simulations on two constructs that isolate the principal stabilizing interactions—the blocked head-free head interface and the blocked head-S2 interface—we introduced physiologically relevant destabilizing factors including mutations, regulatory light chain phosphorylation, high KCl and 2’-deoxy-ADP. This design enabled us to probe conformational heterogeneity and capture the sequence of structural events leading to departure from the IHM state. Our simulations provide unprecedented molecular detail of departure from IHM exit, revealing how specific perturbations alter protein-protein interfaces and shift the balance between inactive and active states. This model indicates that the head-head and head-S2 interfaces present energetically distinct conformational barriers to departure from the IHM; the head-head interface is predicted to be the stronger stabilizer of the IHM state. Further our simulations show that departure from the IHM state requires coordination between the motor domains and the light chain binding regions of the lever arm. Ongoing experiments leverage free energy calculations to predict the conformational changes for IHM departure for a wild type IHM construct. These findings advance the mechanistic understanding of myosin regulation, highlight structural features that can be targeted by small molecules, and establish a framework for modeling transitions into and out of the IHM conformation.
Childers et al. (Sun,) studied this question.