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Regulatory Mechanism of Muscle Contraction using Electron cryo-microscopy and NMR spectroscopy

使用电子冷冻显微镜和核磁共振波谱研究肌肉收缩的调节机制

基本信息

批准号：
15370069
负责人：
WAKABAYASHI Takeyuki
金额：
$ 9.66万
依托单位：
School of Science and Engineering, Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Troponin and tropomyosin on actin filaments constitute a Ca^<2+>-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within actin-based thin filament. Troponin consists of three subunits : an inhibitory subunit (TnI), a Ca^<2+>-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca^<2+>-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT_2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with ^<15>N and/or ^<13>C. By applying NMR spectroscopy, the solution structures of a ‘mobile' actin-binding domain (〜6.1 kDa) in the troponin ternary complex (〜52 kDa) were determined. The mobile domain appears … More to tumble independently of the core domain of troponin. Ca^<2+>-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca^<2+>. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-EM density map of thin filament at low Ca^<2+>. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at low Ca^<2+>. The resultant atomic model suggests that troponin electrostatically interacted with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at low Ca^<2+>. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch. Less

肌动蛋白丝上的肌钙蛋白和原肌球蛋白构成了一个Ca^2+敏感的开关，通过肌动蛋白细丝内的一系列构象变化来调节脊椎动物横纹肌的收缩。肌钙蛋白由三个亚基组成：抑制亚基（TnI）、Ca^2+结合亚基（TnC）和原肌球蛋白结合亚基（TnT）。钙离子与肌钙蛋白C的结合被认为会减弱肌钙蛋白和肌动蛋白之间的相互作用，并引发肌钙蛋白复合物的大的构象变化。然而，肌钙蛋白肌动蛋白结合位点的原子细节尚未确定。从重组鸡骨骼肌肌钙蛋白I、肌钙蛋白C和肌钙蛋白2（TnT的C端区域）中重组出了三元肌钙蛋白复合物，其中只有肌钙蛋白I被均一地标记了^<15>N和/或^<13>C。通过应用核磁共振波谱法，确定了肌钙蛋白三元复合物（约52 kDa）中“移动的”肌动蛋白结合结构域（约6.1 kDa）的溶液结构。出现移动的域 ...更多信息独立于肌钙蛋白的核心结构域。Ca^<2+>引起的化学位移和线形的变化表明，在高Ca^<2+>时，其翻滚受到更大的限制。肌动蛋白和肌钙蛋白的移动的结构域之间相互作用的原子细节是通过将移动的结构域对接到低Ca^<2+>时细丝的冷冻电镜密度图中来定义的。这允许确定TnI的残基133的3D位置，这是结合可用信息的重要标志。这使得肌钙蛋白的整个球状头部区域在低Ca^<2+>时能够独特地对接到细丝冷冻电镜图中。由此产生的原子模型表明，肌钙蛋白与肌动蛋白静电相互作用，并导致原肌球蛋白的移动，以实现肌肉松弛。一个重要的特征是肌钙蛋白的卷曲螺旋区在低Ca^<2+>时推动原肌球蛋白。此外，肌球蛋白与肌动蛋白丝上的移动的结构域之间的关系表明后者充当故障安全闩锁。少