The C terminus of cardiac troponin I stabilizes the Ca2+-activated state of tropomyosin on actin filaments

Abstract

RATIONALE: Ca(2+) control of troponin-tropomyosin position on actin regulates cardiac muscle contraction. The inhibitory subunit of troponin, cardiac troponin (cTn)I is primarily responsible for maintaining a tropomyosin conformation that prevents crossbridge cycling. Despite extensive characterization of cTnI, the precise role of its C-terminal domain (residues 193 to 210) is unclear. Mutations within this region are associated with restrictive cardiomyopathy, and C-terminal deletion of cTnI, in some species, has been associated with myocardial stunning. OBJECTIVE: We sought to investigate the effect of a cTnI deletion-removal of 17 amino acids from the C terminus- on the structure of troponin-regulated tropomyosin bound to actin. METHODS AND RESULTS: A truncated form of human cTnI (cTnI(1-192)) was expressed and reconstituted with troponin C and troponin T to form a mutant troponin. Using electron microscopy and 3D image reconstruction, we show that the mutant troponin perturbs the positional equilibrium dynamics of tropomyosin in the presence of Ca(2+). Specifically, it biases tropomyosin position toward an "enhanced C-state" that exposes more of the myosin-binding site on actin than found with wild-type troponin. CONCLUSIONS: In addition to its well-established role of promoting the so-called "blocked-state" or "B-state," cTnI participates in proper stabilization of tropomyosin in the "Ca(2+)-activated state" or "C-state." The last 17 amino acids perform this stabilizing role. The data are consistent with a "fly-casting" model in which the mobile C terminus of cTnI ensures proper conformational switching of troponin-tropomyosin. Loss of actin-sensing function within this domain, by pathological proteolysis or cardiomyopathic mutation, may be sufficient to perturb tropomyosin conformation.