Structure of the Actin-Tropomyosin-TNT Complex

Abstract

Thin filament-linked proteins actin, tropomyosin, and troponin are the major regulators of cardiac and skeletal muscle that activate contraction through calcium-mediated conformational changes. Single point mutations within the cardiac forms of these proteins cause hypertrophic (HCM) and dilated (DCM) cardiomyopathy, affecting more than 1 in every 500 people. In order to determine mechanisms of thin filament regulation and the initial mutation-associated insults that arise during disease development, it is necessary to define the molecular interactions of the thin filament components. The TnT tail (TnT1), and more specifically the N-terminal region (residues 1-156 in cardiac TnT isoform 6), tethers the troponin core domain (i.e. TnT2, TnC and TnI) to tropomyosin on the thin filament and thereby assumes the 40 nm tropomyosin periodicity. Additionally, TnT1 stabilizes the tropomyosin head-to-tail domain to form cables spanning the full filament. TnT1-156also contains more than one third of all thin filament mutations that cause HCM and DCM. Because no high-resolution structure of this region is available, residue-level interactions with tropomyosin as well as the positions of many HCM and DCM point mutants remain elusive. We are using cryo-electron microscopy to solve the high-resolution structure of the cardiac actin-tropomyosin-TnT1-156 complex. The complex has been fully optimized and 2-dimensional class averages display clear TnT density on actin-tropomyosin. 3-dimensional reconstructions reveal that TnT1-156 makes the tropomyosin appear broader and flatter, consistent with an α-helix binding alongside tropomyosin, rather than hovering over it. Given current cryo-EM optimization at nanometer resolution, we anticipate resolution to near-atomic levels, thus leading to the residue-level determination of the TnT-tropomyosin interaction and a more precise understanding of mutation-associated deficits.