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首页> 外文期刊>Journal of Molecular Biology >Solution structure of the catalytic domain of the mitochondrial protein ICT1 that is essential for cell vitality.
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Solution structure of the catalytic domain of the mitochondrial protein ICT1 that is essential for cell vitality.

机译：线粒体蛋白ICT1催化域的溶液结构，对细胞活力至关重要。

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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 beta1-beta2-alpha1-beta3-alpha2 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 (alpha1), instead of a beta-turn, is inserted between strand beta2 and strand beta3. A characteristic groove formed between alpha1 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.
机译：据报道，ICT1蛋白是人线粒体的组成部分，并通过其保守的GGQ基序具有不依赖密码子的肽基-tRNA水解活性，尽管对其详细机理了解甚少。在这里，我们使用NMR光谱法，确定了小鼠ICT1蛋白催化结构域的溶液结构，该蛋白缺乏N端线粒体靶向信号和无结构的C端富含碱性残基的延伸，并研究了ICT1敲低的作用流式细胞仪检测HeLa细胞中的线粒体（由小干扰RNA介导）。包含206个残基的全长蛋白质残基69-162的催化结构域形成具有beta1-beta2-alpha1-beta3-alpha2拓扑结构的结构和类似于包含GGQ的1类释放域3的结构的结构框架因素（RF）。结构的一半（包括含GGQ的环）具有与RF中相同的序列和结构，这与ICT1对线粒体上ICT1的肽基-tRNA水解活性一致，这类似于RF。但是，该结构的另一半与RF域3的相应部分在形状上有所不同，因为在ICT1中，α-螺旋（alpha1）代替了β-转角，插入了链beta2和链beta3之间。通过基于结构的比对，在alpha1和三链反平行β-折叠之间形成的特征性凹槽被确定为ICT1特定的功能位点。此外，在ICT1中，识别RF中终止密码子的结构域被富含C端基本残基的扩展所取代。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。流式细胞仪分析表明，敲低ICT1会导致凋亡细胞死亡，并降低线粒体膜电位和质量。此外，与对照细胞相比，ICT1敲低细胞的细胞色素c氧化酶活性降低了35％。这些结果表明，ICT1功能对于细胞活力和线粒体功能至关重要。

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