Solution Structure of the Catalytic Domain of the Mitochondrial Protein ICT1 That Is Essential for Cell Vitality
Solution Structure of the Catalytic Domain of the Mitochondrial Protein ICT1 That Is Essential for Cell Vitality
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DOI：
10.1016/j.jmb.2010.09.033
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发表时间：
2010-11-26
影响因子：
5.6
通讯作者：
Nameki, Nobukazu
中科院分区：
文献类型：
--
作者：
Handa, Yoshihiro;Hikawa, Yusuke;Nameki, Nobukazu
ICT1蛋白最近被报道是人类线粒体的一个组成部分，并通过其保守的GGQ基序具有密码子非依赖性的肽基-tRNA水解活性，尽管对详细的机制知之甚少。在这里，使用NMR光谱，我们确定了小鼠ICT1蛋白的催化结构域的溶液结构，缺乏N-末端线粒体靶向信号和非结构化的C-末端碱性残基丰富的延伸，我们研究了ICT1敲低（由小干扰RNA介导）对HeLa细胞线粒体的影响，使用流式细胞术。包含206个残基的全长蛋白质的残基69 - 162的催化结构域形成具有β 1-β 2-α 1-β 3-α 2拓扑结构和类似于1类释放因子（RF）的含GGQ结构域3的结构的结构框架的结构。一半的结构，包括GGQ环，具有基本相同的序列和结构的RF中，与ICT1的肽基-tRNA水解活性的线粒体，这是类似于RF。然而，该结构的另一半在形状上与RF结构域3的相应部分不同，因为在ICT 1中，α-螺旋（α 1）而不是β-转角插入链β 2和链β 3之间。通过基于结构的比对，α 1和三链反向平行β折叠之间形成的特征性凹槽被鉴定为推定的ICT1特异性功能位点。此外，识别RF中终止密码子的结构域在ICT1中被C-末端富含碱性残基的延伸所取代。这些差异似乎与ICT1的特定功能有关，而不是由RF介导的翻译终止。流式细胞术分析表明，ICT1的敲低导致凋亡细胞死亡，线粒体膜电位和质量下降。此外，ICT1敲低细胞中的细胞色素c氧化酶活性比对照细胞中的细胞色素c氧化酶活性降低35%。这些结果表明，ICT1功能是细胞活力和线粒体功能所必需的。(C)2010爱思唯尔有限公司版权所有。
The ICT1 protein was recently reported to be a component of the human mitoribosome and to have codon-independent peptidyl-tRNA hydrolysis activity via its conserved GGQ motif, although little is known about the detailed mechanism. Here, using NMR spectroscopy, we determined the solution structure of the catalytic domain of the mouse ICT1 protein that lacks an N-terminal mitochondrial targeting signal and an unstructured C-terminal basic-residue-rich extension, and we examined the effect of ICT1 knockdown (mediated by small interfering RNA) on mitochondria in HeLa cells using flow cytometry. The catalytic domain comprising residues 69-162 of the 206-residue full-length protein forms a structure with a beta 1-beta 2-alpha 1-beta 3-alpha 2 topology and a structural framework that resembles the structure of GGQ-containing domain 3 of class 1 release factors (RFs). Half of the structure, including the GGQ-containing loop, has essentially the same sequence and structure as those in RFs, consistent with the peptidyl-tRNA hydrolysis activity of ICT1 on the mitoribosome, which is analogous to RFs. However, the other half of the structure differs in shape from the corresponding part of RF domain 3 in that in ICT1, an alpha-helix (alpha 1), instead of a beta-turn, is inserted between strand beta 2 and strand beta 3. A characteristic groove formed between alpha 1 and the three-stranded antiparallel beta-sheet was identified as a putative ICT1-specific functional site by a structure-based alignment. In addition, the structured domain that recognizes stop codons in RFs is replaced in ICT1 by a C-terminal basic-residue-rich extension. It appears that these differences are linked to a specific function of ICT1 other than the translation termination mediated by RFs. Flow cytometry analysis showed that the knockdown of ICT1 results in apoptotic cell death with a decrease in mitochondrial membrane potential and mass. In addition, cytochrome c oxidase activity in ICT1 knockdown cells was decreased by 35% compared to that in control cells. These results indicate that ICT1 function is essential for cell vitality and mitochondrial function. (C) 2010 Elsevier Ltd. All rights reserved.