Yan Wei, Ye Qing, Tan Min, Chen Xi, Eriani Gilbert, Wang En-Duo
From the State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
the College of Life Sciences, Wuhan University, 299 Bayi Road, Wuhan 430072, Hubei, China.
J Biol Chem. 2015 May 8;290(19):12256-67. doi: 10.1074/jbc.M115.639492. Epub 2015 Mar 27.
A conserved structural module following the KMSKS catalytic loop exhibits α-α-β-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA(Leu), which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA(Leu) D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS.
在Ia类和Ib类氨酰-tRNA合成酶中，紧跟KMSKS催化环的一个保守结构模块呈现出α-α-β-α拓扑结构。然而，该结构域的功能鲜受关注。在此，我们通过对来自不同物种的关键残基进行表征，描述了该模块对亮氨酰-tRNA合成酶（LeuRS）的氨酰化和编辑能力的影响。该结构域第三个α-螺旋上高度保守的碱性残基发生突变，会损害LeuRS对tRNA（Leu）反密码子茎的亲和力，从而降低氨酰化和编辑活性。该α-螺旋上的两个甘氨酸残基有助于大肠杆菌LeuRS（EcLeuRS）的灵活性、亮氨酸活化和编辑。β-链上的酸性残基增强了EcLeuRS的编辑活性，并感知tRNA（Leu）D环的大小。引入这些残基可刺激与EcLeuRS的连接多肽1编辑结构域和亮氨酸特异性结构域融合的嵌合简约型支原体移动LeuRS的tRNA依赖性编辑活性。总之，这些结果表明茎接触折叠是催化位点和tRNA结合结构域之间的功能和结构连接体。将EcLeuRS茎接触折叠结构域与编辑缺陷型酶进行序列比较表明，该模块的关键残基已经进化出一种适应策略来适应LeuRS的编辑功能。
