Aaron and the Hoskins Lab at UW Madison (@uwmadisonrna.bsky.social)

Force-Induced Ankle Opening Reveals Mechanical Stabilization of the Ankle of Human β-Cardiac Myosin Human β-cardiac myosin drives contraction in the heart. Extensive biophysical and single-molecule studies have quantified myosin’s chemo-mechanical cycle, which generates ∼5 nm of displacement and 5–7 pN of force. Myosin’s 9 nm-long, α-helical lever arm is rigidified by bound essential and regulatory light chains (ELC and RLC). Numerous pathogenic mutations and sequence-conservation patterns within the lever arm where the RLC binds (LARLC) belie the overly simplified view that the lever arm acts solely as a rigid rod that transduces ATP hydrolysis into motion. Structural studies have shown that myosin adopts an interacting-heads motif (IHM), which inhibits motor activity and mechanically strains the RLC complex, consisting of the RLC bound to the LARLC. Alteration in the configuration of the RLC complex’s “ankle”─a sharp kink in the lever arm─is hypothesized to modulate the propensity of myosin to enter the IHM. To investigate the complex’s mechanical stability, we developed a single-molecule atomic-force microscopy assay with three different pulling geometries: pulling across the LARLC, the RLC, and the RLC complex. When pulling across the LARLC by applying force to its N and C termini, the mechanical dissociation of the RLC was resolved along with two intermediates. Coarse-grained Brownian dynamics detailed these molecular configurations as the opening of myosin’s ankle and the preferential dissociation of one of the RLC’s two EF-hand domains. Moreover, the linker between the EF-hand domains forms an interface with an RLC N-terminal loop. This interface stabilized the native acute ankle angle against opening. Pulling across the RLC and the RLC complex revealed different unfolding pathways, each with one intermediate. Looking forward, these assays can probe for the effects of pathogenic mutations and phosphorylation on the nanomechanics of the RLC complex. https://pubs.acs.org/doi/10.1021/acsnano.5c22720