Recent advances in cardiac myosin research have revealed two relaxed states: the high energy-consuming disordered-relaxed (DRX) state and the low-ATPase super-relaxed (SRX) state. The SRX state, a crucial OFF conformation, is thought to correlate with the interacting-head motif (IHM), in which the S1 heads fold back onto the proximal S2 tail. Assuming that S2 dimerization provides the structural basis for this motif, resolving how the dimer forms and interacts with S1 is essential for understanding myosin regulation. We investigated the stability of the proximal S2 coiled-coil structure and its interaction with the S1 head of human β-cardiac myosin. Four single-cysteine mutants distributed across the N-terminal, C-terminal, and internal regions of S2 were engineered to probe dimerization. Circular dichroism confirmed that the cysteine substitutions did not perturb secondary structure. Microscale thermophoresis and FRET revealed strong and consistent dimer formation for all mutants. We examined how S1 interacts with both monomeric and dimeric forms of S2 to determine whether S2 dimerization influences the accessibility of S1 binding sites and contributes to stabilization of the OFF state. Together, our study establishes a framework for quantifying S2 dimerization and defining its role in S1-S2 interactions. By integrating CD, MST, and FRET, we provide new insights into how the structural dynamics of the S2 dimer influence conformational transitions underlying human β-cardiac myosin function.
Naskar et al. (Sun,) studied this question.