Modeling Human Cardiac Thin Filament Structures

Abstract

Striated muscle contraction is regulated in a calcium-dependent manner through dynamic motions of the tropomyosin/troponin polymer, a multicomponent complex wrapped around actin-containing thin filaments. Tropomyosin/troponin sterically blocks myosin-binding at low-calcium concentrations but moves to expose myosin-binding sites at high-calcium concentrations leading to force development. Understanding the key intermolecular interactions that define these dynamic motions will promote our understanding of mutation-induced contractile dysfunction that eventually leads to hypertrophic cardiomyopathy, dilated cardiomyopathy, and skeletal myopathies. Advancements in cryoelectron microscopy (cryoEM) have resulted in a partial elucidation of structures of the thin filament, revealing many atomic-level interactions between the component proteins and critical calcium-dependent conformational alterations. However, building models at the resolutions achieved can be challenging since landmarks in the maps are often missing or ambiguous. Therefore, current computational analyses including de novo structure prediction, protein-protein docking, molecular dynamics flexible fitting, and molecular dynamics simulations are needed to ensure good quality models. We review here our efforts to model the troponin T domain spanning the head-to-tail overlap domain of tropomyosin, improving previous models. Next, we refined the published cryoEM modeled structures, which had mistakenly compressed alpha helices, with a model that has expected helical parameters while matching densities in the cryoEM volume. Lastly, we used this model to reinterpret the interactions between tropomyosin and troponin I showing key features that hold the tropomyosin cable in its low-calcium, sterically blocking position. These revised thin filament models show improved intermolecular interactions in the key low- and high-calcium regulatory states, providing novel insights into function.

Keywords: actin; cryoEM; protein-protein docking; tropomyosin; troponin.

FIGURE 1

Superposition of the three structural states of tropomyosin during thin filament activation of the M-state (green), C-state (yellow), and B-state (blue) showing the movement of tropomyosin over actin (white surface, pointed end up). Note the steric blocking of the myosin binding site (red) is relieved as tropomyosin moves from B- to M-state.

FIGURE 2

(A) Electron density map of the cardiac thin filament solved in calcium-free conditions. Note the tubular density for both tropomyosin (red, yellow, blue, and purple ribbon) and TnT1 (green ribbon), which could be fit by a number of potential structures. The only landmark here for the modeler is the N- and C-termini of tropomyosin (arrow). (B) Ca²⁺-bound model (Yamada et al., 2020) showing poor alignment of positive (blue sticks) and negative residues (red sticks) between actin (gray ribbon) and tropomyosin (blue and purple ribbons). (C) Ca²⁺-bound model (Pavadai et al., 2020) in a similar orientation as (B). Note the improved alignment of charged residues after rebuilding.