Identification of the Binding Site of the Quinone-Head Group in Mitochondrial Coq10 by Photoaffinity Labeling
Identification of the Binding Site of the Quinone-Head Group in Mitochondrial Coq10 by Photoaffinity Labeling
复制标题
通过光亲和标记鉴定线粒体 Coq10 中醌头基团的结合位点
DOI：
10.1021/bi500347s
复制
发表时间：
2014
期刊：
影响因子：
2.9
通讯作者：
Miyoshi H.
中科院分区：
文献类型：
--
作者：
Murai M;Matsunobu K;Kudo S;Ifuku K;Kawamukai M;Miyoshi H.
线粒体辅酶q10是一种泛素（UQ）结合蛋白，是类固醇急性调节蛋白（StAR）相关脂质转移（START）结构域超家族的成员。Coq10基因的缺失在酿酒酵母（saccharomyces cerevisiae）和pombe裂糖酵母（schizosaccharomyces pombe）中引起明显的呼吸缺陷，这表明Coq10可能支持呼吸复合物之间有效的电子转移；然而，其生理作用仍然难以捉摸。为了阐明辅酶q10的作用，我们试图确定UQ在重组体中的结合位点。pombeCoq10通过光反应性UQ探针UQ-1进行光亲和标记，并通过所谓的点击化学对标记肽进行生物素化，在大肠杆菌细胞膜上表达。综合蛋白质组学分析表明，uq -1的醌头环特异性结合Coq10的Phe39-Lys45的n端区域，该区域与许多含有START结构域的蛋白质的配体结合袋相对应。在存在过量的人工短链UQ类似物（如UQ2）时，标记完全被抑制。在Phe39Ala和Pro41Ala突变体中，标记度分别为野生型Coq10的40%和60%。虽然Coq10一直被认为与UQ结合，但我们的工作首次提供了Coq10在UQ的START结构域容纳醌头环的直接证据。在此基础上，对辅酶q10的生理作用进行了探讨。
Mitochondrial Coq10 is a ubiquinone (UQ)-binding protein that is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain superfamily. Deletion of theCOQ10gene was previously shown to cause a marked respiratory defect inSaccharomyces cerevisiaeandSchizosaccharomyces pombe, which indicated that Coq10 may support efficient electron transfer between the respiratory complexes; however, its physiological role remains elusive. To elucidate the role of Coq10, we attempted to identify the binding site of UQ in recombinantS. pombeCoq10 expressed in anEscherichia colicell membrane through photoaffinity labeling with the photoreactive UQ probe, UQ-1, in combination with biotinylation of the labeled peptide by means of the so-called click chemistry. Comprehensive proteomic analyses revealed that the quinone-head ring of UQ-1specifically binds to the N-terminal region of Phe39–Lys45 of Coq10, which corresponds to the ligand-binding pocket of many proteins containing the START domain. The labeling was completely suppressed in the presence of an excess amount of artificial short-chain UQ analogues, such as UQ2. In the Phe39Ala and Pro41Ala mutants, the extents of labeling were ∼40 and ∼60%, respectively, of that of wild-type Coq10. While Coq10 has been thought to bind UQ, our work first provides the direct evidence of Coq10 accommodating the quinone-head ring of UQ in its START domain. On the basis of these results, the physiological role of Coq10 has been discussed.