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

Abstract

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.

Hypertrophic Cardiomyopathy in Underrepresented Populations: Clinical and Genetic Landscape Based on a Russian Single-Center Cohort Study

“…The THB is a specific motif for the cardiac isoform of MYBP-C, providing the platform for potential binding to actin and myosin [46]. In vitro studies have shown that the mutations in THB can alter these interactions [47]. Further evidence for the causality of the p.R346H variant was obtained by our family studies.…”

“…There were no homozygous or compound heterozygous patients with two truncating variants (Figure 1). Compared to patients with a single P/LP/VUS-LP variant (n = 70), patients with two rare variants (n = 10) had a significantly higher SCD risk score (5.9 [3.5-7.3] vs. 2.9 [1.9-4.0], p = 0.011), larger left atrium (52 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57] vs. 43 [39][40][41][42][43][44][45][46][47][48][49][50] mm, p = 0.017), more frequent high systolic pulmonary artery pressure (63 vs. 14%, p = 0.004), and a trend toward more frequent severe NYHA class III/IV (38 vs. 11%, p = 0.07). The clinical course of HCM was highly variable, ranging from mild symptoms to severe HF progression and SCD in middle age.…”