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(Circulation Research. 2002;91:704.)
© 2002 American Heart Association, Inc.

Molecular Medicine

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

Johanna Moolman-Smook, Emily Flashman, Willem de Lange, Zhili Li, Valerie Corfield, Charles Redwood, Hugh Watkins

From the US/MRC Centre for Molecular and Cellular Biology (J.M.-S., W.d.L., V.C.), University of Stellenbosch, Tygerberg, South Africa; and the Department of Cardiovascular Medicine (E.F., Z.L., C.R., H.W.), University of Oxford, Oxford, UK. Present address for Z.L. is the Department of Cardiology, Fourth Military Medical University, Xi’an City, People’s Republic of China.

Correspondence to Hugh Watkins, MD, PhD, Dept of Cardiovascular Medicine, John Radcliffe Hospital, Oxford, UK OX28AF. E-mail hugh.watkins{at}cardiov.ox.ac.uk

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 >7x10⁶ 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 (K_a=1x10⁵ 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.

Key Words: cardiac myosin binding protein-C • hypertrophic cardiomyopathy • missense mutations • ligand binding

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