Human Cardiac Myosin-Binding Protein C N-Terminal Domains Cooperatively Impact Actin Structural Dynamics

Abstract

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein that modulates contractility by influencing interactions between myosin and actin. Despite being a leading cause of familial hypertrophic cardiomyopathy, the mechanism by which cMyBP-C modulates contractility is not well understood. N-terminal domains of cMyBP-C (C0 through C2) have been shown to interact with the actin-thin filament; however, the cMyBP-C regions involved in the interaction and how cMyBP-C influences actin's dynamic structure remain unclear. Here, we have used cosedimentation assays for measuring binding kinetics and time-resolved phosphorescence anisotropy (TPA) for measuring actin rotational dynamics. We examined the effects of actin interactions with human fragments C0-C1 and C0-C2 of cMyBP-C. We also evaluated roles of cMyBP-C phosphorylation sites known to influence contractility. We find that C0-C2 binds actin with ∼4-fold increase in apparent affinity compared to C0-C1. TPA of labeled-actin showed marked differences in the potency of effects between C0-C2, C0-C1, and phosphorylated cMyBP-C fragments with respect to decreasing the amplitude of torsional motions, increasing the rate of monomer rotation, and increasing relative resilience of the filament. A Hill equation was used to determine the level of cooperativity of the TPA effects for each fragment. Our results demonstrate that C0-C2 exhibits stronger cooperativity in propagating its effects on amplitude, rate, and resilience to more distant actin monomers than C0-C1. Assuming a thin filament cooperative unit of ∼7 actin monomers (length of tropomyosin), our TPA analyses suggest that one molecule of C0-C2 bound to actin propagated effects 2-3 cooperative units (14-21 monomers), whereas C0-C1 effects were confined to one unit, and phosphorylation reduced cooperativity. These findings provide new insight into the molecular basis for cMyBP-C effects on actin structure and function. This work was supported by an NIH R00HL122397 to B.A.C.