Identification of Novel Interactions Between Domains of Myosin Binding Protein-C That Are Modulated by Hypertrophic Cardiomyopathy Missense Mutations

Abstract— Cardiac myosin binding protein-C (cMyBPC) is a modular protein consisting of 11 domains whose precise function and sarcomeric arrangement are incompletely understood. Identification of hypertrophic cardiomyopathy (HCM)–causing missense mutations in cMyBPC has highlighted the significance of certain domains. Of particular interest is domain C5, an immunoglobulin-like domain with a cardiac-specific insert, which is of unknown function yet is the site of two HCM-causing missense mutations. To identify interactors with this region, a human cardiac cDNA library was screened in a yeast two-hybrid (Y2H) assay using the C5 sequence as bait. Screening >7×106 clones surprisingly revealed that domain C5 preferentially bound to clones encoding C-terminal fragments of cMyBPC; the interacting region was narrowed to domain C8 by deletion mapping. A surface plasmon resonance assay using purified recombinant cMyBPC domains was used to measure the affinity of C5 and C8 in vitro (Ka=1×105 mol/L−1). This affinity was decreased about 2-fold by the HCM mutation R654H, and by at least 10-fold by the mutation N755K. Further Y2H assays also demonstrated specific binding between domains C7 and C10 of cMyBPC. Based on these novel interactions, and previous biochemical and structural data, we propose that cMyBPC molecules trimerize into a collar around the thick filament, with overlaps of domains C5-C7 of one cMyBPC with C8-C10 of another. We speculate that this interaction may be dynamically formed and released, thereby restricting or favoring cross-bridge formation, respectively. We suggest that the HCM mutations act by altering the cMyBPC collar, indicating its importance in thick filament structure and regulation.