Mutations in Cardiac Myosin Binding Protein - C Associated with Hypertrophic Cardiomyopathy Alter Structure, F-Actin Binding and Phosphorylation

Abstract

A common cause of hypertrophic cardiomyopathy (HCM) is mutations and modifications of the sarcomeric protein cardiac myosin binding protein C (MyBPC). MyBPC binds to the F-actin thin filament via its N-terminus, the myosin thick filament backbone via its C-terminus, and reversibly binds to the myosin-S2 neck region (when de-phosphorylated). The functional consequences of nine hypertrophic cardiomyopathy mutations within the N-terminal phosphorylation region, domains C1C2 were determined. Of the nine HCM mutations investigated, only two (Tyr237Ser and His257Pro) showed decreased solubility and altered folding, compared to the wild-type C1C2, as measured by circular dichroic spectroscopy. Phosphorylation did not alter the secondary structure content of C1C2. Wild-type C1C2 binds F-actin with 1:1 stoichiometry and micromolar affinity (Kd ∼ 7 μM), using a co-sedimentation assay. All mutants exhibited weaker binding to actin, except for Asp228Asn and Arg326Gln, which bound similarly to WT. Protein kinase A phosphorylation activity was assessed in four HCM mutants, located in close proximity to the phosphorylation sites. The Gly278Glu mutation inhibited phosphorylation, Arg326Gln and Leu352Pro increased the rate, while no difference was observed for the Gly279Ala mutation. The order of protein kinase A phosphorylation of C1C2 was determined to be: phosphorylation of Ser284 (Site B), followed by Ser275 (Site A). The third and final phosphate was added at Ser304 (Site C), at a much slower rate (0.015 min 1). The effects of hypertrophic cardiomyopathy mutations on C1C2 structure and function were consistent with the mild phenotype associated with these mutations.