Ueki Shoji, Nakamura Motoyoshi, Komori Tomotaka, Arata Toshiaki
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Biochemistry. 2005 Jan 11;44(1):411-6. doi: 10.1021/bi048110w.
Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.
肌钙蛋白C（TnC）氨基末端结构域中钙诱导的结构转变触发骨骼肌和心肌收缩。这种结构转变的显著特征是B螺旋和C螺旋的移动，这被称为N结构域的“打开”。这种移动暴露出一个疏水区域，使得能够与肌钙蛋白I（TnI）的调节结构域相互作用，如在肌钙蛋白三元复合物的晶体结构中所见[武田，S.，山下，A.，前田，K.，和前田，Y.（2003年）《自然》424，35 - 41]。与骨骼肌TnC不同，钙结合位点I（一个由A螺旋 - 环 - B螺旋基序组成的EF手基序）在心肌TnC中无活性。与骨骼肌TnC比较产生的问题是，在心肌TnC中，两个螺旋如何根据钙结合移动或与TnI相互作用。在本研究中，我们通过定点自旋标记电子顺磁共振（EPR）研究了在心肌TnC单体状态和TnC - TnI二元复合物中，B螺旋和C螺旋相对于D螺旋的钙诱导移动。制备了双自旋标记的TnC突变体，并通过傅里叶反卷积方法分析偶极相互作用来估计自旋 - 自旋距离。在镁饱和单体状态下，对于在B螺旋上的G42C和D螺旋上的C84处自旋标记的突变体，估计自旋间距离为18.4埃。在相同条件下，C螺旋上的Q58C和D螺旋上的C84之间的自旋间距离估计为18.3埃。通过添加钙离子和与TnI形成复合物观察到距离变化。我们的数据表明，C螺旋以明显的钙依赖方式远离D螺旋，而B螺旋没有。观察到B螺旋通过与TnI相互作用而移动。还表明钙和TnI对于心肌TnC中N结构域的完全打开也是必不可少的。
