Abstract

Mutations in the cardiac thin filament (TF) have highly variable effects on the regulatory function of the cardiac sarcomere. Understanding the molecular-level dysfunction elicited by TF mutations is crucial to elucidate cardiac disease mechanisms. The hypertrophic cardiomyopathy-causing cardiac troponin T (cTnT) mutation Δ160Glu (Δ160E) is located in a putative "hinge" adjacent to an unstructured linker connecting domains TNT1 and TNT2. Currently, no high-resolution structure exists for this region, limiting significantly our ability to understand its role in myofilament activation and the molecular mechanism of mutation-induced dysfunction. Previous regulated in vitro motility data have indicated mutation-induced impairment of weak actomyosin interactions. We hypothesized that cTnT-Δ160E repositions the flexible linker, altering weak actomyosin electrostatic binding and acting as a biophysical trigger for impaired contractility and the observed remodeling. Using time-resolved FRET and an all-atom TF model, here we first defined the WT structure of the cTnT-linker region and then identified Δ160E mutation-induced positional changes. Our results suggest that the WT linker runs alongside the C terminus of tropomyosin. The Δ160E-induced structural changes moved the linker closer to the tropomyosin C terminus, an effect that was more pronounced in the presence of myosin subfragment (S1) heads, supporting previous findings. Our in silico model fully supported this result, indicating a mutation-induced decrease in linker flexibility. Our findings provide a framework for understanding basic pathogenic mechanisms that drive severe clinical hypertrophic cardiomyopathy phenotypes and for identifying structural targets for intervention that can be tested in silico and in vitro.

Highlights

  • Mutations in the cardiac thin filament (TF) have highly variable effects on the regulatory function of the cardiac sarcomere

  • Proteolytic studies demonstrated that cardiac troponin T (cTnT) comprises two functional domains: the Tm-binding TNT1 domain and the calcium-sensitive TNT2 domain, which binds cardiac troponin I, cardiac troponin C, and Tm [8, 9]

  • This limited structural information on the cTnT linker region greatly impairs our ability to understand the mechanism of TF activation and molecular pathogenesis of HCM induced by mutations in this region

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Summary

Introduction

Mutations in the cardiac thin filament (TF) have highly variable effects on the regulatory function of the cardiac sarcomere. A long (ϳ50-amino acid (aa)) flexible linker connecting the TNT1 and TNT2 domains (Fig. 1A) is essential for transmitting the calcium-induced conformational changes from the C-terminal end of cTnT to the N terminus. CD studies suggested that the TNT1 domain and the proximal linker region have an ␣-helical character [10]; the structural details of these functionally important domains were unknown because of their absence from the crystal structure of the human cTn core [11] and cryo-EM reconstruction of the cardiac TF [12] This limited structural information on the cTnT linker region greatly impairs our ability to understand the mechanism of TF activation and molecular pathogenesis of HCM induced by mutations in this region. The reported statistics are the results of a one-way ANOVA with Sidak correction. ****, p Ͻ 0.0001 versus A168C; ####, p Ͻ 0.0001 versus A177C; ††††, p Ͻ 0.0001 versus A192C

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