BPS2026 – Structural dynamics of myosin loop 2 as a regulator of actomyosin interaction

For muscles to contract, the motor protein myosin must bind to actin and initiate cross-bridge cycling. Our recent work has identified the loop 2 domain of myosin as an important regulator of actomyosin interaction. We showed that by adopting distinct spatial conformations, loop 2 strongly influences myosin’s affinity for actin; however, the mechanisms by which these conformations are formed, stabilized, or regulated remain unknown. Here, we build upon our previous findings to show that, despite its intrinsically disordered nature, loop 2 transiently adopts secondary structure elements that stabilize specific loop 2 conformations, which in turn correlate with varying levels of actin-myosin binding affinity. To probe this mechanism, we combined molecular dynamics simulations of myosin with electrostatic calculations to investigate how loop 2 structural transitions shape actomyosin interactions. We found that loop 2 has a propensity to form ɑ-helical hydrogen bonds, thereby assuming a retracted conformation that sequesters its positive charges and markedly decreases actin affinity. Conversely, formation of β-sheet hydrogen bonds stabilized an unfurled state that enhanced actin affinity. In addition to wild-type myosin, we examined cardiomyopathy-associated mutations E525K, V606M, R369Q, A223T, and G256E, which are known to perturb loop 2. In mutants previously demonstrated to show increased actin binding affinity, the overall propensity to form semi-stable conformations, as measured by RMSD and secondary structure frequency, was preserved across genotypes; however, the types of secondary structures formed varied. In our simulations, mutant myosin exhibited reduced α-helix hydrogen bond formation but increased β-sheet hydrogen bond content, as well as overall enhanced actin affinity. Together, these results demonstrate that myosin’s loop 2 forms transient conformations stabilized by distinct secondary structures, and that these conformations correlate with differential actin affinity, providing a potential structural mechanism for the regulation of actin-myosin binding.