Comparative studies on the functional roles of N‐ and C‐terminal regions of molluskan and vertebrate troponin‐I
Comparative studies on the functional roles of N‐ and C‐terminal regions of molluskan and vertebrate troponin‐I
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DOI：
10.1111/j.1742-4658.2005.04866.x
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发表时间：
2005-09
期刊：
影响因子：
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通讯作者：
Hiroyuki Tanaka;Yuhei Takeya;Teppei Doi;F. Yumoto;M. Tanokura;I. Ohtsuki;K. Nishita;T. Ojima
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文献类型：
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作者：
Hiroyuki Tanaka;Yuhei Takeya;Teppei Doi;F. Yumoto;M. Tanokura;I. Ohtsuki;K. Nishita;T. Ojima
脊椎动物肌钙蛋白通过钙离子依赖的方式将肌钙蛋白I(TnI)与肌动蛋白或肌钙蛋白C(TNC)选择性结合，从而调节肌肉收缩。为了阐明软体动物肌钙蛋白调控的分子机制，我们比较了Akazara扇贝TnI和兔快骨骼TnI重组片段的功能特性。赤潮扇贝TnI的C末端片段(ATnI232−292)，包含TnI抑制区(兔TnI的第104-115个残基)和调节的TnC结合位点(第116-131个残基)，结合了肌动蛋白原肌球蛋白和抑制了肌动蛋白原肌球蛋白镁ATPase。然而，即使在有钙离子存在的情况下，它也不与TNC相互作用。这些结果表明，在软体动物肌钙蛋白中没有观察到参与该区域交替结合的机制。另一方面，ATnI130TnC252含有−的结构结合部位(兔TnI的1-30个残基)和抑制区，与肌动蛋白和TnC都有很强的结合。此外，由该片段、肌钙蛋白-T和TNC组成的三元复合体以钙离子依赖的方式激活ATPase，几乎与完整的Akazara扇贝肌钙蛋白一样有效。因此，Akazara扇贝肌钙蛋白通过激活机制来调节收缩，激活机制涉及从结构TNC结合部位到TnI抑制区域的区域。再加上相应的兔TnI片段(RTnI1TnI116)表现出类似的激活作用，这些发现表明TnIN末端区域不仅对维持肌钙蛋白复合体的结构完整性而且对−+依赖的激活具有重要意义。
Vertebrate troponin regulates muscle contraction through alternative binding of the C‐terminal region of the inhibitory subunit, troponin‐I (TnI), to actin or troponin‐C (TnC) in a Ca2+‐dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C‐terminal fragment of Akazara scallop TnI (ATnI232−292), which contains the inhibitory region (residues 104–115 of rabbit TnI) and the regulatory TnC‐binding site (residues 116–131), bound actin‐tropomyosin and inhibited actomyosin‐tropomyosin Mg‐ATPase. However, it did not interact with TnC, even in the presence of Ca2+. These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI130−252, which contains the structural TnC‐binding site (residues 1–30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin‐T, and TnC activated the ATPase in a Ca2+‐dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC‐binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI‐fragment (RTnI1−116) shows similar activating effects, these findings suggest the importance of the TnI N‐terminal region not only for maintaining the structural integrity of troponin complex but also for Ca2+‐dependent activation.