Structural analysis of human cardiac troponin C and myosin binding protein C

Sarcomeric proteins are essential for the proper structural assembly and functioning of the sarcomere, the basic contractile unit in striated muscles. When mutations are present in the genes that encode for these proteins, it may lead to cardiac diseases, such as hypertrophic cardiomyopathy (HCM), the leading cause of death in young athletes. Hundreds of mutations within the genes that encode sarcomeric proteins have been shown to cause HCM. The L29Q mutation in cardiac troponin C (cTnC) and the R502W mutation in cardiac myosin binding protein C (cMyBP-C) are two of these mutations. cTnC senses the cytosolic Ca2+ concentration and transduces this signal to allow for cross-bridging, leading to muscle contraction. cMyBP-C regulates muscle contractility by interacting with myosin and actin. How the L29Q and the R502W mutations, respectively, affect the cTnC and cMyBP-C and cause disease is unclear. To advance our knowledge of how these two mutations affect the cardiac proteins’ structures and functions, I first generated high resolution structures of the wild type (WT) and mutant regulatory domains of cTnC (cNTnC) using X-ray crystallography and then, determined the WT and R502W mutant structures of the C3 domain of cMyBP-C using nuclear magnetic resonance (NMR). The WT cNTnC was discovered to have coordinating Cd2+ ions at both of its calcium-binding sites. This is true for the mutant cNTnC as well. In the WT cNTnC, the vestigial site (EF1) coordinated Cd2+ in a noncanonical ‘distorted’ octahedral geometry, while the functional calcium-binding site (EF2) coordinated Cd2+ in the canonical pentagonal bipyramidal geometry. A subtle structural change was observed in the region near the L29Q mutation, and it may play a role in the increased Ca2+ affinity of the mutant. The R502W mutation in cMyBP-C did not change the protein’s global structure. The dynamics and thermal stabilities of the protein were also not affected by the mutation, as shown by techniques such as amide 15N relaxation and circular dichroism spectroscopy. The mutation does, however, alter the surface charge on the C3 domain and, like other HCM-related mutations found within the same domain, may disrupt the protein’s interactions with other sarcomeric proteins, such as actin. The data acquired from this thesis project contributes to a better understanding of the structures of sarcomeric proteins and the pathophysiology of hypertrophic cardiomyopathy.