Structural insight into unique cardiac myosin-binding protein-C motif: A partially folded domain

Abstract

The structural role of the unique myosin-binding motif (m-domain) of cardiac myosin-binding protein-C remains unclear. Functionally, the m-domain is thought to directly interact with myosin, whereas phosphorylation of the m-domain has been shown to modulate interactions between myosin and actin. Here we utilized NMR to analyze the structure and dynamics of the m-domain in solution. Our studies reveal that the m-domain is composed of two subdomains, a largely disordered N-terminal portion containing three known phosphorylation sites and a more ordered and folded C-terminal portion. Chemical shift analyses, d NN(i, i + 1) NOEs, and 15N{ 1H} heteronuclear NOE values show that the C-terminal subdomain (residues 315-351) is structured with three well defined helices spanning residues 317-322, 327-335, and 341-348. The tertiary structure was calculated with CS-Rosetta using complete 13Cα, 13Cβ, 13C′, 15N, 1Hα, and 1H N chemical shifts. An ensemble of 20 acceptable structures was selected to represent the C-terminal subdomain that exhibits a novel three-helix bundle fold. The solvent-exposed face of the third helix was found to contain the basic actin-binding motif LK(R/K)XK. In contrast, 15N{ 1H} heteronuclear NOE values for the N-terminal subdomain are consistent with a more conformationally flexible region. Secondary structure propensity scores indicate two transient helices spanning residues 265-268 and 293-295. The presence of both transient helices is supported by weak sequential d NN(i, i + 1) NOEs. Thus, the m-domain consists of an N-terminal subdomain that is flexible and largely disordered and a C-terminal subdomain having a three-helix bundle fold, potentially providing an actin-binding platform. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.