Structure and Dynamics of the Flexible Cardiac Troponin T Linker Domain in a Fully Reconstituted Thin Filament

Abstract

The structural analysis of large protein complexes has been greatly enhanced through the application of electron microscopy techniques. One such multiprotein complex, the cardiac thin filament (cTF), has cyclic interactions with thick filament proteins to drive contraction of the heart that has recently been the subject of such studies. As important as these studies are, they provide limited or no information on highly flexible regions that in isolation would be characterized as inherently disordered. One such region is the extended cardiac troponin T (cTnT) linker between the regions of cTnT which have been labeled TNT1 and TNT2. It comprises a hinge region (residues 158-166) and a highly flexible region (residues 167-203). Critically, this region modulates the troponin/tropomyosin complex's position across the actin filament. Thus, the cTnT linker structure and dynamics are central to the regulation of the function of cardiac muscles, but up to now, it was ill-understood. To establish the cTnT linker structure, we coupled an atomistic computational cTF model with time-resolved fluorescence resonance energy transfer measurements in both ±Ca²⁺ conditions utilizing fully reconstituted cTFs. We mapped the cTnT linker's positioning across the actin filament, and by coupling the experimental results to computation, we found mean structures and ranges of motion of this part of the complex. With this new insight, we can now address cTnT linker structural dynamics in both myofilament activation and disease.

Figure 1.

Structure of the cTF highlighting the conformations of the cTnT linker: Computational model of the human cTF: troponin complex (cTnT: teal; cTnI: pink; cTnC: golden brown); Tm dimer (green and orange); and actin monomer (transparent). (A) Original computational model incorporating Yamada et al.’s refined structural components. (B,C) The computational model was constrained to the measured FRET distances (Table 1) and then allowed to evolve unconstrained to obtain the cTnT short (B) and long (C) linker representations. The black spheres show the locations of cTnT FRET labels. All models are for the +Ca²⁺ condition.

Figure 2.

A168C to S198C Intra-FRET confirms short and long cTnT linker conformations: the FRET donor and acceptor probe locations (black) on the cTF as shown by the computational model (A,C) and schematic representations (B,D). (A,B) represent the short cTnT linker orientation, and (C,D) represent the long cTnT linker orientation. All models are for reference and are in the +Ca²⁺ condition. (E) Intra-FRET measurements in the ±Ca²⁺ conditions were checked for normality via the Shapiro–Wilk test, which demonstrates the shift in data distribution in the +Ca²⁺ condition. **p < 0.01 Shapiro–Wilk test, n = 12 for −Ca²⁺; n = 16 for +Ca²⁺. The subsequent normality test upon mapping FRET distances, based on computational model predictions, showed two normally distributed means in the +Ca²⁺ condition (data not shown).

Figure 3.

cTnT linker and actin FRET donor and acceptor sites mapped to the computationally derived short and long linker conformations: FRET donor (black) and acceptor (red) probe locations on the computational model and schematic of the cTF. (A,B) represents the short cTnT linker orientation and (C,D) represents the long cTnT linker orientation. All models are for the +Ca²⁺ condition.

Figure 4.

cTnT linker-to-actin FRET distance measurements as mapped to the computational model in each biochemical condition: FRET distances mapped to their probable actin monomer are shown in the −Ca²⁺ and +Ca²⁺ condition. Two-way ANOVA was used to analyze the change in each linker site position relative to their neighboring sites and to assess the effect of changing biochemical condition (±Ca²⁺) for each linker site. MCs (Sidak) were used to assess specific differences when a main effect was observed. * vs cTnT 168, # vs cTnT 177, *p < 0.05. Exact p-values are reported in Tables S2 and S3.