Muscle contraction is governed by tropomyosin (Tpm) shifting azimuthally between three states on F-actin (B-, C-, and M-states) in response to calcium binding to troponin and actomyosin cross-bridge formation. The Tpm coiled coil polymerizes head to tail along the long-pitch helix of F-actin to form continuous superhelical cables that wrap around the actin filaments. The end-to-end bonds formed between the N- and C-terminus of adjacent Tpm molecules define Tpm continuity and play a critical role in the ability of Tpm to cooperatively bind to actin, thus facilitating Tpm conformational switching to cooperatively propagate along F-actin. We expect that a missense mutation in this critical overlap region associated with dilated cardiomyopathy, A277V, will alter Tpm binding and thin filament activation by altering the overlap structure. Here, we used cosedimentation assays and in vitro motility assays to determine how the mutation alters Tpm binding to actin and its ability to regulate actomyosin interactions. Analytical viscometry coupled with molecular dynamics simulations showed that the A277V mutation results in enhanced Tpm end-to-end bond strength and a reduced curvature of the Tpm overlap domain. The mutant Tpm exhibited enhanced actin-Tpm binding affinity, consistent with overlap stabilization. The observed A277V-induced decrease in cooperative activation observed with regulated thin filament motility indicates that increased overlap stabilization is not correlated with Tpm-Tpm overlap binding strength or mechanical rigidity as is often assumed. Instead, A277V-induced structural changes result in local and delocalized increases in Tpm flexibility and prominent coiled-coil twisting in pseudorepeat 4. An A277V-induced decrease in Ca2+ sensitivity, consistent with a mutation-induced bolstering of the B-state Tpm-actin electrostatic contacts and an increased Tpm troponin T1 binding affinity, was also observed.

中文翻译：

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心肌病突变改变肌球蛋白的端到端连接并降低钙敏感性

肌肉收缩由原肌球蛋白 (Tpm) 在 F-肌动蛋白的三个状态（B-、C-和 M-状态）之间沿方位角移动来控制，以响应钙与肌钙蛋白的结合和肌动球蛋白跨桥形成。Tpm 盘绕线圈沿着 F-肌动蛋白的长螺距螺旋从头到尾聚合，形成连续的超螺旋电缆，缠绕在肌动蛋白丝周围。相邻 Tpm 分子的 N 端和 C 端之间形成的端到端键定义了 Tpm 的连续性，并在 Tpm 与肌动蛋白协同结合的能力中起关键作用，从而促进 Tpm 构象转换沿 F-协同传播肌动蛋白。我们预计与扩张型心肌病 A277V 相关的这个关键重叠区域中的错义突变将通过改变重叠结构来改变 Tpm 结合和细丝激活。这里，我们使用共沉淀试验和体外运动试验来确定突变如何改变 Tpm 与肌动蛋白的结合及其调节肌动蛋白相互作用的能力。分析粘度计结合分子动力学模拟表明，A277V 突变导致 Tpm 端对端键强度增强和 Tpm 重叠域曲率降低。突变体 Tpm 表现出增强的肌动蛋白-Tpm 结合亲和力，与重叠稳定一致。观察到的 A277V 诱导的协同激活减少与调节的细丝运动性表明增加的重叠稳定性与通常假设的 Tpm-Tpm 重叠结合强度或机械刚度无关。反而，