BPS2026 – The G256E HCM mutation prolongs relaxation via altered nucleotide handling

Genetic mutations in many sarcomeric proteins are linked with familial forms of hypertrophic cardiomyopathy (HCM). Human induced pluripotent stem cell-derived cardiomyocytes have emerged as a powerful system to study the effects of HCM-linked mutations on impaired cardiac performance. Additionally, high-resolution structural models of actin and myosin have resolved major structural changes related to the force-generating steps of the chemo-mechanical cycle. These experimental systems can be used synergistically to model the structure-function origins of cardiac disease resulting from pathogenic sarcomere protein variants. Using an interdisciplinary approach, we analyzed the chemo-mechanical behavior of the myosin variant G256E at multiple scales. At the atomic and molecular scale, molecular dynamics (MD) simulations revealed that G256E induces structural changes in the nucleotide-binding pocket, indicative of an increased affinity of myosin for ADP, which was further validated through stopped-flow biochemistry. Steered MD simulations suggested alterations in the timing and sequence of contact pair disruptions and an increase in the work required to displace ADP·Mg 2+ from the binding pocket. In myofibrils, delayed ADP release manifested as a prolongation and reduced ADP sensitivity of the early phase of relaxation, believed to represent the rate of cross-bridge detachment. These findings highlight the importance of ADP release in the chemo-mechanical cycle and identify the molecular mechanisms by which an HCM-linked mutation leads to disease phenotypes by modifying ADP release. Furthermore, this study demonstrates that small-scale changes in the structure of myosin’s active site can lead to kinetic changes in actomyosin cycling that manifest as elevated force generation and impaired relaxation through large-scale changes in cross-bridge cycling. Overall, our study highlights how contemporary methods can explain not only the phenotypic consequences of HCM mutations but also the underlying structural and biophysical origins of disease.