Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletal muscle troponin-C photo-cross-link to Met121 of troponin-I.
Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletal muscle troponin-C photo-cross-link to Met121 of troponin-I.
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兔骨骼肌肌钙蛋白-C N 末端疏水口袋的残基 48 和 82 与肌钙蛋白-I 的 Met121 光交联。
DOI：
10.1021/bi9824341
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Tao,T
中科院分区：
文献类型：
--
作者：
Luo,Y;Leszyk,J;Qian,Y;Gergely,J;Tao,T
它已经被提出[Herzberg et al. (1986)J。医学杂志。化学，261,2638−2644]，并由结构研究证实[Gagne et al. (1995)Nat。结构体。Biol. 2,784−789]，Ca2+与肌钙蛋白-C （TnC）触发位点的结合导致由B、C和D螺旋结合的n端疏水口袋打开。这种构象变化被认为为肌钙蛋白- i （TnI）提供了一个额外的结合位点，并导致Ca2+调节过程中的进一步事件。为了回答TnI的哪一部分与TnC的疏水斑块相互作用的问题，我们构建了两个TnC突变体，每个突变体都有一个半胱氨酸，一个在B和C螺旋之间的残基48，另一个在D螺旋上的残基82。每个突变体用光激活交联剂二苯甲酮-4-碘乙酰胺标记，然后进行重组和紫外线照射。研究了TnC和TnI组成的二元配合物，TnC、TnI和肌钙蛋白- t （TnT）组成的三元配合物，以及由肌钙蛋白、原肌球蛋白和f -肌动蛋白组成的合成细丝。在两种突变体和所有三种类型的复合物中均观察到TnC - TnI光交联。虽然在二元和三元配合物的光交联中没有Ca2+依赖性，但在没有Ca2+的情况下，交联的程度比在细丝中存在的程度降低。通过蛋白水解后的微测序和质谱分析，从抑制区c端的5个残基TnI Met121被确定为两个TnC突变体的交联位点。在完整的TnC·TnI复合物中获得的这些结果表明，含有Met121的TnI片段与TnC的n端疏水贴片紧密接触，并且在没有Ca2+的情况下，在细丝中含有该残基的片段稍微远离疏水贴片，可能触发TnI的肌动蛋白结合区(s)向肌动蛋白的易位。
It has been proposed [Herzberg et al. (1986)J. Biol. Chem. 261, 2638−2644], and confirmed by structural studies [Gagne et al. (1995)Nat. Struct. Biol. 2, 784−789], that the binding of Ca2+to the triggering sites in troponin-C (TnC) causes the opening of the N-terminal hydrophobic pocket bound by the B, C, and D helices. This conformational change is believed to provide an additional binding site for troponin-I (TnI) and to lead to further events in the Ca2+regulation process. To answer the question of which part of TnI interacts with this hydrophobic patch of TnC, we constructed two TnC mutants, each with a single cysteine, one at residue 48 between helices B and C and the other at residue 82 on the D helix. Each mutant was labeled with the photoactivatable cross-linker benzophenone-4-iodoacetamide, followed by reconstitution and UV irradiation. Studies were made in the binary complex composed of TnC and TnI, the ternary complex composed of TnC, TnI, and troponin-T (TnT), and the synthetic thin filament composed of troponin, tropomyosin, and F-actin. TnC−TnI photo-cross-linking was observed for both mutants and for all three types of complexes. Although no Ca2+dependence in the photo-cross-linking was observed on the binary and ternary complexes, the extent of cross-linking was reduced in the absence vs the presence of Ca2+in the thin filament. TnI Met121, five residues from the C-terminus of the inhibitory region, was identified as the cross-linking site for both TnC mutants using microsequencing and mass spectrometry following proteolysis. These results, obtained with intact TnC·TnI complexes, indicate that the TnI segment containing Met121 is in close contact with the N-terminal hydrophobic patch of TnC, and that in the thin filament the segment containing this residue moves away slightly from the hydrophobic patch in the absence of Ca2+, possibly triggering the translocation of the actin-binding region(s) of TnI toward actin.