Defining the Flexible Cardiac Troponin T Linker Region in Relationship to Actin and Determining the Effects of Pathogenic Point Mutations

Abstract

Contraction of the heart is driven by cyclic interactions between the thick and thin filament proteins, mediated by Ca2+ level fluctuations. Recent advances in electron microscopy (EM) and molecular dynamics studies have provided structural understanding of the cardiac thin filament (cTF). However, for certain cTF domains the structure and precise nature of the inter- and intra-protein interactions remain unknown. One such region is the extended cardiac troponin t (cTnT) linker between TNT1 and TNT2, which remains structurally undefined due to its inherent flexibility. It is comprised of a hinge region (residues 158–166) and a flexible region (residues 166–203), which modulates the cTn-Tm complex’s position on actin, affecting the crossbridge cycling efficiency. The cTnT linker contains two mutational hotspots which cause severe and highly penetrant hypertrophic (HCM) and dilated (DCM) cardiomyopathies. Thus cTnT linker structural alterations could affect both inter- and intra-protein interactions and alter myofilament activation; yet, a refined structural model of the cTF inclusive of the cTnT linker does not exist limiting our understanding of the molecular mechanisms involved. To establish a cTnT linker high resolution structural model an atomistic computational cTF model was coupled with time-resolved fluorescence resonance energy transfer (TR-FRET) measurements in both ±Ca2+ conditions utilizing fully reconstituted cTFs. First, the existence of the two cTnT linker populations, “short” and “long” that had been observed in recent cryo-EM studies were validated. Secondly, the cTnT linker’s structure was positioned across the actin filament. Third, it was determined how cTnT linker point mutations linked to cardiomyopathies altered the wildtype linker structure. For FRET, cTnT linker residues A168, A177, A192, and S198 were sequentially cysteine-substituted and IAEDANS-donor-labeled. The DABMI-acceptor-label was attached to cTnT A168C or S198C to confirm the two cTnT linker conformations and the 5-IAF-acceptor was attached to actin Cys-374 for cTnT linker to actin mapping. The computational cTF model was then constrained to reflect the measured TR-FRET distances and processed to an equilibrated state. The constraints were then removed and the computational cTF model reprocessed to develop refined structures. Using this coupled approach, a cTF high resolution structure that included the refined cTnT linker structure was developed. This new cTF structural model can now provide mechanistic insight into the cTnT linker structural dynamics in both myofilament activation and disease.