Site-Directed Spin Labeling Electron Paramagnetic Resonance Study of the Calcium-Induced Structural Transition in the N-Domain of Human Cardiac Troponin C Complexed with Troponin I

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• 作者：Shoji Ueki ; Motoyoshi Nakamura ; Tomotaka Komori ; and Toshiaki Arata
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：January 11, 2005
• 年：2005
• 卷：44
• 期：1
• 页码：411 - 416
• 全文大小：113K
• 年卷期：v.44,no.1(January 11, 2005)
• ISSN：1520-4995

文摘

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggersskeletal and cardiac muscle contraction. The salient feature of this structural transition is the movementof the B and C helices, which is termed the "opening" of the N-domain. This movement exposes ahydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seenin 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, Ca²⁺-binding site I (an EF-hand motif thatconsists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising fromcomparisons with skeletal TnC is how both helices move according to Ca²⁺ binding or interact with TnIin cardiac TnC. In this study, we examined the Ca²⁺-induced movement of the B and C helices relativeto the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directedspin 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 deconvolutionmethod. An interspin distance of 18.4 Å was estimated for mutants spin labeled at G42C on the B helixand C84 on the D helix in a Mg²⁺-saturated monomer state. The interspin distance between Q58C on theC helix and C84 on the D helix was estimated to be 18.3 Å under the same conditions. Distance changeswere observed by the addition of Ca²⁺ ions and the formation of a complex with TnI. Our data indicatedthat the C helix moved away from the D helix in a distinct Ca²⁺-dependent manner, while the B helix didnot. A movement of the B helix by interaction with TnI was observed. Both Ca²⁺ and TnI were alsoshown to be essential for the full opening of the N-domain in cardiac TnC.