The Effects of C-Terminal Mutations on the Folding of Cardiac Myosin Binding Protein-C

Abstract

Cardiac myosin binding protein-C (cMyBP-C) is a modular protein involved in stabilizing interactions with the thick filament of the sarcomere. The N-terminus of cMyBP-C associates with actin and myosin S2 and the C-terminus interacts with titin and myosin rods. While no high-resolution structure of C-terminal cMyBP-C exists, disruption of this region is proposed to destabilize cMyBP-C and adversely affect cardiac structure and function. In particular, deletion of 25 base pairs (Δ25) in the gene encoding for cMyBP-C results in amino acid substitutions in the C10 domain of cMyBP-C (C10 Δ25) which may be associated with the development of hypertrophic and dilated cardiomyopathies by unknown molecular mechanisms. The prevalence of this mutation is approximately 1% of the world population, underscoring the necessity of determining its role(s) in the pathogenesis of cardiomyopathies. In this study, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies have been used to examine the conformation of wild-type (Wt) and mutant C-terminal domains of cMyBP-C. Comparison of near UV CD spectra revealed alterations in the packing of aromatic residues in C10 Δ25 suggesting it is less stably folded as compared to C10 Wt. C10 Δ25 exhibited less beta-sheet content than C10 Wt as evidenced by the estimation of secondary structure from CD data. NMR analyses of amide proton/nitrogen chemical shifts and line-widths were used to probe the conformation of C10 domains and to map residues of importance in protein-protein association onto cMyBP-C models. Taken together, these data suggest that the Δ25 mutation structurally modulates cMyBP-C sites involved in binding titin and myosin.