Experimental approaches such as fiber diffraction and cryo-electron microscopy reconstruction have defined regulatory positions of tropomyosin on actin but have not, as yet, succeeded at determining key atomic-level contacts between these proteins or fully substantiated the dynamics of their interactions at a structural level. To overcome this deficiency, we have previously employed computational approaches to deduce global dynamics of thin filament components by energy landscape determination and molecular dynamics simulations. Still, these approaches remain computationally challenging for any complex and large macromolecular assembly like the thin filament. For example, tropomyosin cable wrapping around actin of thin filaments features both head-to-tail polymeric interactions and local twisting, both of which depart from strict superhelical symmetry. This produces a complex energy surface that is difficult to model and thus to evaluate globally. Therefore, at this stage of our understanding, assessing global molecular dynamics can prove to be inherently impractical. As an alternative, we adopted a "divide and conquer" protocol to investigate actin-tropomyosin interactions at an atomistic level. Here, we first employed unbiased protein-protein docking tools to identify binding specificity of individual tropomyosin pseudorepeat segments over the actin surface. Accordingly, tropomyosin "ligand" segments were rotated and translated over potential "target" binding sites on F-actin where the corresponding interaction energetics of billions of conformational poses were ranked by the programs PIPER and ClusPro. These data were used to assess favorable interactions and then to rebuild models of seamless and continuous tropomyosin cables over the F-actin substrate, which were optimized further by flexible fitting routines and molecular dynamics. The models generated azimuthally distinct regulatory positions for tropomyosin cables along thin filaments on actin dominated by stereo-specific head-to-tail overlap linkage. The outcomes are in good agreement with current cryo-electron microscopy topology and consistent with long-thought residue-to-residue interactions between actin and tropomyosin.

中文翻译：

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蛋白质-蛋白质对接揭示了原肌球蛋白对肌动蛋白丝的动态相互作用

纤维衍射和冷冻电子显微镜重建等实验方法已经定义了原肌球蛋白对肌动蛋白的调节位置，但尚未成功确定这些蛋白质之间的关键原子级接触或在结构水平上完全证实它们相互作用的动力学. 为了克服这一缺陷，我们以前采用计算方法通过能量景观确定和分子动力学模拟来推断细丝组件的全局动力学。尽管如此，对于像细丝这样的任何复杂和大的大分子组装，这些方法在计算上仍然具有挑战性。例如，缠绕在细丝肌动蛋白周围的原肌球蛋白电缆具有头对尾聚合物相互作用和局部扭曲的特点，两者都背离了严格的超螺旋对称。这会产生一个复杂的能量表面，难以建模，因此难以进行全局评估。因此，在我们理解的这个阶段，评估全球分子动力学可能被证明是不切实际的。作为替代方案，我们采用了“分而治之”的协议，在原子水平上研究肌动蛋白-原肌球蛋白相互作用。在这里，我们首先采用无偏见的蛋白质-蛋白质对接工具来识别肌动蛋白表面上单个原肌球蛋白假重复片段的结合特异性。因此，原肌球蛋白“配体”片段在 F-肌动蛋白上潜在的“目标”结合位点上旋转和翻译，其中数十亿构象姿势的相应相互作用能量由程序 PIPER 和 ClusPro 排序。这些数据用于评估有利的相互作用，然后在 F-肌动蛋白基质上重建无缝和连续的原肌球蛋白电缆模型，并通过灵活的拟合程序和分子动力学进一步优化。这些模型沿着由立体特异性头尾重叠连接主导的肌动蛋白上的细丝生成了原肌球蛋白电缆在方位角上不同的调节位置。结果与当前的冷冻电子显微镜拓扑结构非常一致，并且与肌动蛋白和原肌球蛋白之间长期考虑的残留物相互作用一致。这些模型沿着由立体特异性头尾重叠连接主导的肌动蛋白上的细丝生成了原肌球蛋白电缆在方位角上不同的调节位置。结果与当前的冷冻电子显微镜拓扑结构非常一致，并且与肌动蛋白和原肌球蛋白之间长期考虑的残留物相互作用一致。这些模型沿着由立体特异性头尾重叠连接主导的肌动蛋白上的细丝生成了原肌球蛋白电缆在方位角上不同的调节位置。结果与当前的冷冻电子显微镜拓扑结构非常一致，并且与肌动蛋白和原肌球蛋白之间长期考虑的残留物相互作用一致。