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

肌动蛋白丝上的肌钙蛋白和原肌球蛋白构成一个Ca^<2+>-敏感开关，通过肌动蛋白基细丝内的一系列构象变化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）。Ca^<2+>-结合TnC被认为削弱了肌钙蛋白和肌动蛋白之间的相互作用，并引发了肌钙蛋白复合物的大构象变化。然而，肌钙蛋白的肌动蛋白结合位点的原子细节尚未确定。用重组鸡骨骼TnI、TnC和TnT_2 （TnT的C端区）构建了三元肌钙蛋白复合物，其中只有TnI被统一标记为^<15>N和/或^<13>C。应用核磁共振波谱技术，确定了肌钙蛋白三元配合物（~ 52 kDa）中一个“可移动”的肌动蛋白结合域（~ 6.1 kDa）的溶液结构。移动区域看起来…更多的是独立于肌钙蛋白的核心区域。Ca^<2+>引起的化学位移和线形变化表明，在高Ca^<2+>时，其翻滚更受限制。通过在低Ca^<2+>下将肌动蛋白与肌钙蛋白的移动结构域对接到细丝的低温电镜密度图中，定义了肌动蛋白与肌钙蛋白的移动结构域相互作用的原子细节。这使得确定TnI残基133的三维位置成为纳入现有信息的重要里程碑。这使得肌钙蛋白的整个球形头部区域在低Ca^<2+>的薄丝低温电镜图中实现了独特的对接。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，引起原肌凝蛋白的移位，从而实现肌肉松弛。一个重要的特征是肌钙蛋白的盘绕区将原肌凝蛋白推至低Ca^<2+>。此外，肌凝蛋白和肌动蛋白丝上的移动结构域之间的关系表明，后者作为一个故障安全锁存器。少

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