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.

Figure 1. Electron micrographs of reconstituted thin filaments

(a) Bare actin filaments. (b) Actin filaments reconstituted with wild-type troponin and tropomyosin in Ca²⁺-free buffer. (c) Actin filaments reconstituted with wild-type troponin and tropomyosin in the presence of Ca²⁺. (d) Actin filaments reconstituted with mutant troponin and tropomyosin in Ca²⁺-free buffer. (e) Actin filaments reconstituted with mutant troponin and tropomyosin in the presence of Ca²⁺. Arrowheads point to the globular heads of the troponin complex. The white arrows highlight tropomyosin strands. Scale bar = 50 nm.

Figure 2. Mutant troponin affects the average position of tropomyosin on actin

3D reconstructions of filaments are depicted in longitudinal view. (b–d) show 3D-structures of filaments incubated in Ca²⁺ whereas (e–g) depict reconstructions of Ca²⁺-free filaments. The bare actin filament is depicted in (a). Subdomains 1 and 2 comprise the outer domain of actin (A_o) whereas the inner domain (A_i) consists of subdomains 3 and 4. Panel (b) depicts actin-tropomyosin with wild-type troponin in Ca²⁺. Panel (c) depicts of mutant-controlled tropomyosin on actin in Ca²⁺. In (d) the positions of tropomyosin from (b) and (c) are superimposed. Panels (e) and (f) show the average position of tropomyosin conferred by wild-type and mutant troponin, respectively, in the absence of Ca²⁺. The results of (e) and (f) are superimposed in (g). All structures are superimposed in (h).

Figure 3. Cross-sections of wild-type- and mutant-controlled thin filaments

Panels correspond to those described in figure 2. Structures in Ca2+ (b–d); structures in EGTA (e–h); wild-type + Ca²⁺ (b); mutant + Ca²⁺ (c); panels (b) and (c) are superimposed in (d). wild-type in EGTA (e); mutant in EGTA (f); panels (e) and (f) are superimposed in (g). All structures are superimposed in (h)

Figure 4. Helical projections illustrate the impact of mutant troponin on the tropomyosin position

(a) Actin-tropomyosin-troponin filaments in Ca²⁺-free solution. Tropomyosin sits on the outer domain of actin. (b) In Ca²⁺, tropomyosin adopts an average position over the inner domain of actin. (c) Mutant Troponin containing cTnI_1–192, is also responsive to Ca²⁺ and tropomyosin again adopts and average position over the inner domain of actin. (d) Superimposing results from (b) and (c) shows that tropomyosin, controlled by mutant troponin, has shifted azimuthally to adopt an average position further from the outer actin domain by about 9°. (e) To better visualize the image densities arising from the tropomyosin strands, the image density of bare actin filaments was subtracted from (b) and (c). The densities of tropomyosin controlled by wild-type (light green) or mutant troponin (dark green) were superimposed over the helical projection of bare actin (blue). The difference in the centroid positions of tropomyosin density were statistically significant at >95%.