Docking Troponin T onto the Tropomyosin Overlapping Domain of Thin Filaments

Abstract

Complete description of thin filament conformational transitions accompanying muscle regulation requires ready access to atomic structures of actin-bound tropomyosin-troponin. To date, several molecular-docking protocols have been employed to identify troponin interactions on actin-tropomyosin because high-resolution experimentally determined structures of filamentassociated troponin are not available. However, previously published all-atom models of the thin filament show chain separation and corruption of components during our molecular dynamics simulations of the models, implying artifactual subunit organization, possibly due to incorporation of unorthodox tropomyosin-TnT crystal structures and complex FRET measurements during model construction. For example, the recent Williams et al. ( 2016) atomistic model of the thin filament displays a paucity of salt bridges and hydrophobic complementarity between the TnT tail (TnT1) and tropomyosin, which is difficult to reconcile with the high, 20 nM K d binding of TnT onto tropomyosin. Indeed, our molecular dynamics simulations show the TnT1 component in their model partially dissociates from tropomyosin in under 100 ns, whereas actin-tropomyosin and TnT1 models themselves remain intact. We therefore revisited computational work aiming to improve TnT1-thin filament models by employing unbiased docking methodologies, which test billions of trial rotations and translations of TnT1 over three-dimensional grids covering end-to-end bonded tropomyosin alone or tropomyosin on F-actin. We limited conformational searches to the association of well-characterized TnT1 helical domains and either isolated tropomyosin or actin-tropomyosin yet avoided docking TnT domains that lack known or predicted structure. The docking programs PIPER and ClusPro were used, followed by interaction energy optimization and extensive molecular dynamics. TnT1 docked to either side of isolated tropomyosin but uniquely onto one location of actin-bound tropomyosin. The antiparallel interaction with tropomyosin contained abundant salt bridges and intimately integrated hydrophobic networks joining TnT1 and the tropomyosin N-/C-terminal overlapping domain. The TnT1-tropomyosin linkage yields well-defined molecular crevices. Interaction energy measurements strongly favor this TnT1-tropomyosin design over previously proposed models.

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

“…This cryo-EM study of the cardiac thin filament achieved resolution sufficient to create atomic models including most of troponin, in both the presence and absence of Ca 2+ . The structural findings were supported and extended throughout the year by excellent independent reports ( Pavadai et al, 2020b ; Pavadai et al, 2020a ; Oda et al, 2020 ; Doran et al, 2020 ). At the resolution pertinent to the broad themes in our study, these other studies either agree with Yamada et al (2020) or disagree only in the sense of omission (fewer parts of troponin were resolved).…”

“…The largest mutation density within TnT, 15.6%, was in the TnT 87–150 helix that anchors troponin on the thin filament ( White et al, 1987 ; Hinkle et al, 1999 ; Gangadharan et al, 2017 ; Palm et al, 2001 ; Jin and Chong, 2010 ) and overlies the end-to-end connections between adjacent tropomyosins ( Yamada et al, 2020 ; White et al, 1987 ; Pavadai et al, 2020b ; Murakami et al, 2008 ). Several HCM- or DCM-causing causing mutations have been studied in great detail after being discovered in this region and in the evolutionarily conserved preceding few residues in the TnT sequence ( Watkins et al, 1995b ; Tardiff, 2011 ; Mirza et al, 2005 ; Gollapudi et al, 2015 ; Manning et al, 2012 ; McConnell et al, 2017 ).…”

“…The structure of troponin as bound to the actin filament was derived, in both the presence and absence of Ca 2+ . Although the results may be challenged or revised in time, independent cryo-EM or computational work ( Doran et al, 2020 ; Oda et al, 2020 ; Pavadai et al, 2020a ; Pavadai et al, 2020b ; Burbaum et al, 2020 Preprint ; Wang et al, 2020 Preprint ) is already extending or confirming many aspects, such as the orientation of the troponin core domain. These recent developments are transformative for structural understanding of the primary on-off regulatory switch in cardiac and other striated muscles: reversible Ca 2+ binding to troponin.…”

M8R tropomyosin mutation disrupts actin binding and filament regulation: The beginning affects the middle and end

“…The overlap of ;11 residues of each terminus of neighboring tropomyosin dimers form a four-helix bundle (7). The N terminus of the troponin subunit T forms a complex with this head-to-tail four-helix bundle of tropomyosin (8,9), offering additional stabilization (10,11), which slows myosin-ATPase activity (12) when assessed in vitro. The C terminus of troponin T and the troponin complex subunits I and C bind both to the central portion of the tropomyosin dimer and to actin (9,13,14).…”

Complexity in genetic cardiomyopathies and new approaches for mechanism-based precision medicine

“…The troponin complex core domain was crystalized in 2003 ( Takeda et al, 2003 ); however, the majority of troponin T, including R92, was not resolved, likely because this region is intrinsically disordered in solution. Recently, a high-resolution cryo-EM structure of the thin filament provided near-atomic resolution structures of actin, tropomyosin, and most of the troponin complex ( Yamada et al, 2020 ), and the position of troponin T was further refined by computational docking ( Pavadai et al, 2020 ). The majority of troponin T forms an elongated structure, and R92 is located near the region of troponin T that interacts with tropomyosin.…”