Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.
Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.
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DOI：
10.1074/jbc.m110.120014
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发表时间：
2010-10-22
期刊：
影响因子：
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通讯作者：
Lécine P
中科院分区：
文献类型：
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作者：
Lugari A;Betzi S;Decroly E;Bonnaud E;Hermant A;Guillemot JC;Debarnot C;Borg JP;Bouvet M;Canard B;Morelli X;Lécine P
SARS冠状病毒蛋白质组中的几种蛋白质-蛋白质相互作用已被鉴定，其中之一是非结构蛋白nsp 10和nsp 16之间的相互作用。在这项工作中，我们绘制了参与这种相互作用的nsp 10表面上的关键残基。丙氨酸扫描诱变、生物信息学和分子建模被用于鉴定几个“热点”，如Val 42、Met 44、Ala 71、Lys 93、Gly 94和Tyr 96，它们形成约830 μ 2的连续蛋白质-蛋白质表面，携带冠状病毒中非常保守的氨基酸。因为nsp 16仅在其相互作用配偶体nsp 10存在时携带RNA帽2′-O-甲基转移酶（2′O-MTase）活性（Bouvet，M.，德巴诺角，澳-地英伯特岛，Selisko，B.，Snijder，E. J.，Canard，B.，和Decroly，E.（2010）PLoS Pathog. 6，e1000863），在该表面上的突变的功能后果进行了生物化学评价。大多数破坏nsp 10-nsp 16相互作用而不影响结构的变化都能消除对nsp 16 RNA帽2′O-MTase活性的刺激。更引人注目的是，Y 96 A突变消除了对nsp 16 2′O-MTase活性的刺激，而Y 96 F过度刺激了它。因此，nsp 10-nsp 16界面可能是抗人类和动物病原性冠状病毒的抗病毒药物的一个有吸引力的靶点。
Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.