Identification of IscU residues critical for de novo iron-sulfur cluster assembly
Identification of IscU residues critical for de novo iron-sulfur cluster assembly
复制标题
鉴定对铁硫簇从头组装至关重要的 IscU 残基
DOI：
10.1111/mmi.14392
复制
发表时间：
2019
影响因子：
3.6
通讯作者：
Takahashi Yasuhiro
中科院分区：
文献类型：
--
作者：
Tanaka Naoyuki;Yuda Eiki;Fujishiro Takashi;Hirabayashi Kei;Wada Kei;Takahashi Yasuhiro
IscU是ISC机制的核心组成部分，并作为铁-硫（Fe-S）簇在其递送至靶向apo-Fe-S蛋白之前重新组装的支架。然而，分子机制尚未完全理解。本研究对E. coliIscU使用最近开发的突变体的遗传互补系统，该突变体可以在没有Fe-S簇的情况下生存。铁硫簇配体（C37，C63，H105，C106）和近端D39和K103残基是IscU体内功能所必需的，不能被任何其他氨基酸取代。此外，我们还发现，在IscU同源物中严格保守的残基Y3的取代，在体内功能被取消。令人惊讶的是，IscS中的第二位点抑制突变（A349 V）恢复了由IscU Y3取代引起的缺陷。生化分析表明，IscU Y3是至关重要的功能相互作用与IscS和硫转移之间的两种蛋白质。我们的研究结果表明，IscU Y3的关键作用与IscS的柔性环的构象动力学有关，这是巧妙的硫转移到IscU所必需的。
IscU is a central component of the ISC machinery and serves as a scaffold for thede novoassembly of iron–sulfur (Fe–S) clusters prior to their delivery to target apo‐Fe–S proteins. However, the molecular mechanism is not yet fully understood. In this study, we have conducted mutational analysis ofE. coliIscU using the recently developed genetic complementation system of a mutant that can survive without Fe–S clusters. The Fe–S cluster ligands (C37, C63, H105, C106) and the proximal D39 and K103 residues are essential forin vivofunction of IscU and could not be substituted with any other amino acids. Furthermore, we found that substitution of Y3, a strictly conserved residue among IscU homologs, abolishedin vivofunctions. Surprisingly, a second‐site suppressor mutation in IscS (A349V) reverted the defect caused by IscU Y3 substitutions. Biochemical analysis revealed that IscU Y3 was crucial for functional interaction with IscS and sulfur transfer between the two proteins. Our findings suggest that the critical role of IscU Y3 is linked to the conformational dynamics of the flexible loop of IscS, which is required for the ingenious sulfur transfer to IscU.