Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.
Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.
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DOI：
10.1371/journal.ppat.1002059
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发表时间：
2011-05
期刊：
影响因子：
6.7
通讯作者：
Canard B
中科院分区：
文献类型：
--
作者：
Decroly E;Debarnot C;Ferron F;Bouvet M;Coutard B;Imbert I;Gluais L;Papageorgiou N;Sharff A;Bricogne G;Ortiz-Lombardia M;Lescar J;Canard B
细胞和病毒S-腺苷蛋氨酸依赖的甲基转移酶参与代谢、解毒、信号转导、染色质重塑、核酸加工和mR NA封顶等多种调控过程。严重急性呼吸综合征冠状病毒NSP16蛋白是一种S-腺苷蛋氨酸依赖(核苷-2‘-O)甲基转移酶，只有在其激活伙伴NSP10存在的情况下才具有活性。我们报道了NSP10/NSP16复合结构，它显示NSP10通过NSP10中的∼930ä2表面积与NSP16结合。功能分析确定了nsp10/nsp16结合中的关键残基，以及RNA结合或催化中的关键残基，后者可能通过SN2类机制。我们还提出了另外两种晶体结构，即S-腺苷蛋氨酸结合口袋中的抑制剂新芬净和更紧密的复合体NSP10(Y96F)/NSP16，这为(+)RNA病毒中RNA帽酶的调控提供了第一个结构洞察力。2003年出现了一种新的冠状病毒，并被确认为这种名为严重急性呼吸综合征的致命疾病的病原体。这种冠状病毒使用16种非结构蛋白(nsp1-16)复制和转录其巨型基因组。病毒RNA是有上限的，以确保稳定性、高效翻译和避开宿主细胞的天然免疫系统。Nsp16蛋白是一种RNA帽修饰酶，只有在其激活伙伴nsp10存在的情况下才有活性。我们已经结晶了nsp10/16络合物，并以原子分辨率报道了它的晶体结构。NSP10通过NSP10中的RNA930？2活化表面积与NSP16结合，生成的复合体具有∼帽(核苷-2‘-O)-甲基转移酶活性。我们已经进行了突变和功能分析，以确定参与催化和/或RNA结合的关键残基，以及nsp10与nsp16的结合。我们提供了两个额外的晶体结构，一个是已知的SAM结合口袋中结合的抑制剂Sinefunin的晶体结构，另一个是由突变体nsp10(Y96F)与nsp16结合形成的更紧密的复合体。我们的研究为抗病毒药物的设计提供了基础，也为(+)RNA病毒中RNA封闭酶的调控提供了第一个结构性的见解。
Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Å2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses. A novel coronavirus emerged in 2003 and was identified as the etiological agent of the deadly disease called Severe Acute Respiratory Syndrome. This coronavirus replicates and transcribes its giant genome using sixteen non-structural proteins (nsp1-16). Viral RNAs are capped to ensure stability, efficient translation, and evading the innate immunity system of the host cell. The nsp16 protein is a RNA cap modifying enzyme only active in the presence of its activating partner nsp10. We have crystallized the nsp10/16 complex and report its crystal structure at atomic resolution. Nsp10 binds to nsp16 through a ∼930 Å2 activation surface area in nsp10, and the resulting complex exhibits RNA cap (nucleoside-2′-O)-methyltransferase activity. We have performed mutational and functional assays to identify key residues involved in catalysis and/or in RNA binding, and in the association of nsp10 to nsp16. We present two additional crystal structures, that of the known inhibitor Sinefungin bound in the SAM binding pocket, and that of a tighter complex made of the mutant nsp10(Y96F) bound to nsp16. Our study provides a basis for antiviral drug design as well as the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.