A weak coupling mechanism mediates the recovery stroke of myosin VI: a free energy simulation and string method analysis
Authors: Blanc Florian E.C. , Houdusse Anne , Cecchini Marco 
Published: October 2023

Myosin motors use the energy of ATP to produce force and directed movement on actin by a swing of the lever arm. ATP is hydrolysed during the off-actin re-priming transition termed recovery stroke. To provide an understanding of chemo-mechanical transduction by myosin, it is critical to determine how the reverse swing of the lever arm and ATP hydrolysis are coupled. Previous studies concluded that the recovery stroke of myosin II is initiated by closure of the Switch II loop in the nucleotide-binding site. Recently, we proposed that the recovery stroke of myosin VI starts with the spontaneous re-priming of the converter domain to a putative pre-transition state (PTS) intermediate that precedes Switch II closing and ATPase activation. Here, we investigate the transition from the pre-recovery, post-rigor (PR) state to PTS in myosin VI using geometric free energy simulations and the string method. First, our calculations rediscover the PTS state agnostically and show that it is accessible from PR via a low free energy transition path. Second, separate path calculations using the string method illuminate the mechanism of the PR to PTS transition with atomic resolution. In this mechanism, the initiating event is a large movement of the converter/lever-arm region that triggers rearrangements in the Relay-SH1 region and the formation of the kink in the Relay helix with no coupling to the active site. Analysis of the free-energy barriers along the path suggests that the converter-initiated mechanism is much faster that the one initiated by Switch II closure, which supports the biological relevance of PTS as a major on-pathway intermediate of the recovery stroke in myosin VI. Our analysis suggests that lever-arm re-priming and ATP hydrolysis are only weakly coupled, so that the myosin recovery stroke is mostly driven by thermal fluctuations and stabilised by nucleotide consumption via a ratchet-like mechanism.

Author summary

Myosin is an ATP-powered motor protein that is crucial for cellular functions like muscle contraction, cell division, and the transport of molecular cargos. Understanding how myosin transforms chemical energy from ATP hydrolysis into mechanical work is a central open question in structural bioenergetics, which could guide the design of next-generation synthetic nanomachines. In myosin, ATP hydrolysis is coupled to the re-priming of the lever-arm during the recovery stroke transition. Using advanced molecular dynamics simulations, we described the sequence of events and the energetics of the recovery stroke of myosin VI with an unprecedented level of detail. The results support a mechanism in which the re-priming of the mechanical amplifier region of the protein is almost entirely driven by thermal fluctuations and stabilized by ATP hydrolysis. This weakly-coupled mechanism suggests that myosin can “rectify” thermal fluctuations to work efficiently in an isothermal environment dominated by stochastic fluctuations like the cell.