The HCM Mutation L348P in cMyBP-C Enhances Thin Filament Activation through Tropomyosin Shift

Abstract

Cardiac myosin binding protein C (cMyBP-C) is a thick filament protein that plays an important role in modulating contraction of the heart. Mutations in cMyBP-C are a major cause of inherited hypertrophic cardiomyopathy (HCM). In addition to binding to myosin, cMyBP-C also interacts with thin filaments via its N-terminal region, mainly the C1 and M-domains, enhancing thin filament Ca2+-sensitivity. L348P is an HCM-causing mutation that occurs in a conserved sequence in the M-domain, enhancing cMyBP-C binding to actin and thin filament Ca2+-sensitization (Bezold et al., JBC 2013). Our goal was to elucidate the structural basis of L348P's gain of function. By EM 3D reconstruction we previously showed that binding of N-terminal cMyBP-C fragments to actin causes displacement of tropomyosin from its blocked (low Ca2+) position to its closed (high Ca2+) position (Mun et al., PNAS 2014), consistent with the increase in thin filament Ca2+-sensitivity. Phosphorylation of the M-domain resulted in a smaller movement of tropomyosin. Here we have investigated the impact of the L348P mutation on tropomyosin shift. Thin filaments were decorated with wild type C1C2 (containing the C1, C2 and M-domains) and with C1C2 having the L348P mutation. F-actin reconstructions showed additional density on actin subdomain 1 with both fragments, but the density was larger with L348P. When thin filaments at low Ca2+ were decorated with C1C2, tropomyosin moved from the blocked to the closed position, as found previously. In contrast, C1C2-L348P showed a significantly larger tropomyosin shift, to approximately the open position, consistent with L348P's greater Ca2+-sensitization of motility and enhanced Ca2+ sensitivity of tension in cardiac myocytes. Preliminary studies showed a smaller shift with phosphorylated than with unphosphorylated C1C2, while phosphorylated C1C2-L348P showed a larger shift, comparable to L348P.