N-terminal cardiac myosin-binding protein C interactions with myosin and actin filaments using time-resolved FRET

Myosin binding protein-C (cMyBP-C) is a sarcomeric protein responsible for normal contraction and relaxation of the heart. We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to resolve the interactions of cardiac myosin and F-actin with cMyBP-C, focusing on the N-terminal region. The results imply roles of these bound protein complexes in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure and hypertrophic cardiomyopathy (HCM). N-terminal cMyBP-C domains C0 through C2 (C0-C2) contain binding regions for interactions with both thick (myosin) and thin (actin) filaments. Phosphorylation by protein kinase A (PKA) in the cMyBP-C motif (M-domain) regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C and either myosin or actin. We engineered intermolecular pairs of labeling sites between donor-labeled myosin regulatory light chain (V105C) or F-actin (C374) and cMyBP-C (S85C in C0, C249 in C1, or P330C in M-domain) to detect interactions. Phosphorylation reduced the interaction of cMyBP-C to both myosin and actin. Further insight was gained from evaluating cMyBP-C HCM mutations T59A, R282W, E334K, and L349R, which revealed increases in myosin-FRET, increases or decreases in actin-FRET, and perturbations of phosphorylation effects. These findings elucidate binding of cMyBP-C to myosin or actin under physiological and pathological conditions, providing new molecular insight into the modulatory role of these protein-protein interactions in cardiac muscle contractility. Further, these findings suggest that the TR-FRET assays are suitable for rapid and accurate determination of quantitative binding for screening physiological conditions and compounds that affect cMyBP-C interactions with myosin or F-actin for therapeutic discovery. Significance Statement Hypertrophic cardiomyopathy (HCM) is a heritable heart disease involving mutations in genes encoding cardiac muscle proteins. Investigating the underlying molecular mechanisms of HCM mutations provides critical insight into the clinical outcomes and can translate into life-saving therapies. A leading cause of inherited HCM are mutations found in cardiac myosin binding protein-C (cMyBP-C), which binds to both myosin and actin to finely-tune contractility. Efforts in elucidating the details of cMyBP-C interactions with myosin and actin have been limited due to standard techniques that are low-throughput and labor-intensive. We have developed a set of Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays that report the phosphorylation-sensitive binding of N-terminal cMyBP-C to myosin or actin in a high-throughput plate reader format. We detect altered binding due to phosphorylation and unique changes in HCM mutant cMyBP-C binding to myosin versus actin. Our results are informative for developing precision medicine screening assays and new therapies for HCM. ### Competing Interest Statement B.A.C. serves as President of BC Biologics LLC. This relationship has been reviewed and managed by the University of Arizona. BC Biologics had no role in this study. B.A. Colson and F.L. Wong filed a provisional patent application (patent pending, application no. 63/076,735) and B.A. Colson filed a patent application (patent pending, application no. PCT/US21/14142) based on this work. The other authors declare no competing financial interests.