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 filament-associated 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 Kd 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.

中文翻译：

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对接肌钙蛋白-T 到细丝的原肌球蛋白重叠域

伴随肌肉调节的细丝构象转变的完整描述需要随时访问肌动蛋白结合的原肌球蛋白-肌钙蛋白的原子结构。迄今为止，已经采用了几种分子对接协议来识别肌动蛋白-原肌球蛋白上的肌钙蛋白相互作用，因为无法获得高分辨率实验确定的丝状肌钙蛋白结构。然而，之前发表的细丝的全原子模型在我们对模型的分子动力学模拟过程中显示出链分离和组分的损坏，这意味着人为的亚基组织，可能是由于在模型期间加入了非正统的原肌球蛋白-TnT 晶体结构和复杂的 FRET 测量建造。例如，最近的威廉姆斯等人。(2016) 细丝的原子模型显示 TnT 尾部 (TnT1) 和原肌球蛋白之间缺乏盐桥和疏水互补性，这很难与 TnT 与原肌球蛋白的 20 nM Kd 高结合相协调。事实上，我们的分子动力学模拟显示，他们模型中的 TnT1 成分在 100 ns 内与原肌球蛋白部分分离，而肌动蛋白原肌球蛋白和 TnT1 模型本身保持完整。因此，我们重新审视了旨在通过采用无偏对接方法来改进 TnT1 细丝模型的计算工作，该方法测试了 TnT1 在三维网格上的数十亿次试验旋转和平移，覆盖了单独的端到端结合的原肌球蛋白或 F-肌动蛋白上的原肌球蛋白。我们将构象搜索限制为充分表征的 TnT1 螺旋结构域与分离的原肌球蛋白或肌动蛋白原肌球蛋白的关联，但避免对接缺乏已知或预测结构的 TnT 结构域。使用对接程序 PIPER 和 ClusPro，然后进行相互作用能量优化和广泛的分子动力学。TnT1 停靠在分离的原肌球蛋白的任一侧，但唯一地位于肌动蛋白结合的原肌球蛋白的一个位置。与原肌球蛋白的反平行相互作用包含丰富的盐桥和紧密集成的疏水网络，连接 TnT1 和原肌球蛋白 N-/C-末端重叠域。TnT1-原肌球蛋白连接产生明确的分子裂缝。与先前提出的模型相比，相互作用能量测量强烈支持这种 TnT1 原肌球蛋白设计。