Crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp15 suggests a role for hexamerization as an allosteric switch
Crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp15 suggests a role for hexamerization as an allosteric switch
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DOI：
10.1128/jvi.02817-06
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发表时间：
2007-06-01
影响因子：
5.4
通讯作者：
Kuhn, Peter
中科院分区：
文献类型：
--
作者：
Joseph, Jeremiah S.;Saikatendu, Kumar Singh;Kuhn, Peter
来自严重急性呼吸综合征冠状病毒（SARS-CoV）的成熟非结构蛋白-15 （nsp15）含有一种新的尿苷酸特异性Mn2+依赖核糖核酸内切酶（NendoU）。对两种冠状病毒（SARS-CoV和小鼠肝炎病毒）的专性六聚体酶的全长形式及其单体同源物非洲爪蟾（xenou）的结构研究，结合诱变研究表明，酶活性和n端结构域的几个残基是六聚体化的主要决定因素。然而，在NendoUs中六聚化和酶活性之间的紧密联系仍然是一个谜。在这里，我们报告了一个修剪过的、单体形式的SARS-CoV nsp15（残基28至335）的结构，确定了2.9埃的分辨率。催化环（残基234至249）及其两个活性组氨酸（His 234和His 249）被戏剧性地弯曲了大约120度，形成活性位点裂缝。此外，催化亲核试剂lys289指向完全相反的方向，这是支撑环（残基276至295）向外移位的结果。在全长六聚体形式中，这两个环相互堆积，并通过密切的亚基相互作用稳定下来。我们的研究结果支持了一个假设，即由于六聚体结构域的缺失而导致邻近单体的缺失是活性位点破坏的最可能原因，这为为什么只有六聚体形式的酶是有活性的提供了结构基础。
Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn2+-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 angstrom. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically Hipped by similar to 120 degrees into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.