Interaction of cardiac troponin C and troponin I with W7 in the presence of three functional regions of cardiac troponin I
Interaction of cardiac troponin C and troponin I with W7 in the presence of three functional regions of cardiac troponin I
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DOI：
10.1021/bi060779a
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发表时间：
2006-08-15
期刊：
影响因子：
2.9
通讯作者：
Sykes, Brian D.
中科院分区：
文献类型：
--
作者：
Li, Monica X.;Hoffman, Ryan M. B.;Sykes, Brian D.
W7是一种众所周知的钙调蛋白（CaM）拮抗剂，被认为是肌钙蛋白c介导的心肌收缩Ca2+激活的抑制剂。在这项研究中，我们使用核磁共振光谱研究了W7与游离心肌肌钙蛋白C （cTnC）或与心肌肌钙蛋白I （cTnI）肽复合物的结合。用W7滴定cTnC， 3Ca（2+）表明，整个序列的残基，包括cTnC的N-和c -结构域以及中心连接体，都受到了影响。结合化学计量学和化学位移变化轨迹分析表明，W7的结合发生在多个位点。为了解决多位点结合是否与肌钙蛋白复合物相关的问题，W7被滴定为cTnC-cTnI复合物(cTnC, 3Ca(2+), cTnI（34-71), cTnI(128-163)）。在cTnI的N端（类似于34-71的残基）、抑制区（类似于128-147的残基）和开关区（类似于147-163的残基）存在的情况下，W7只会引起N-结构域的化学位移变化，而不会引起c -结构域或cTnC的中心连接体的化学位移变化。结果表明，当cTnI存在时，W7不再与cTnC的多个位点结合，而是特异性结合到N-结构域，并且这种结合（K-D = 0.5 +/- 0.1 mM）可以与cTnI的开关区一起发生。因此，W7可能在直接调节cTnC调控域Ca2+敏感性以及cTnI和cTnC开关区的相互作用中发挥作用。
W7 is a well-known calmodulin (CaM) antagonist and has been implicated as an inhibitor of the troponin C-mediated Ca2+ activation of cardiac muscle contraction. In this study, we use NMR spectroscopy to study binding of W7 to cardiac troponin C (cTnC) free or in complex with cardiac troponin I (cTnI) peptides. Titration of cTnC, 3Ca(2+) with W7 shows that residues throughout the sequence, including the N- and C-domains of cTnC and the central linker, are affected. Analysis of the binding stoichiometry and the trajectories of chemical shift changes indicate that W7 binding occurs at multiple sites. To address the issue of whether multiple-site binding is relevant within the troponin complex, W7 is titrated to a cTnC-cTnI complex (cTnC, 3Ca(2+), cTnI(34-71), cTnI(128-163)). In the presence of the N- terminal ( residues similar to 34-71), inhibitory (residues similar to 128-147), and switch (residues similar to 147-163) regions of cTnI, W7 induces chemical shift changes only in the N- domain and not in the C-domain or the central linker of cTnC. The results indicate that in the presence of cTnI, W7 no longer binds to multiple sites of cTnC but instead binds specifically to the N- domain, and the binding (K-D = 0.5 +/- 0.1 mM) can occur together with the switch region of cTnI. Hence, W7 may play a role in directly modulating the Ca2+ sensitivity of the regulatory domain of cTnC and the interaction of the switch region of cTnI and cTnC.