Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.
Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.
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DOI：
10.1073/pnas.2106379119
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发表时间：
2022-03-01
影响因子：
11.1
通讯作者：
Aihara H
中科院分区：
文献类型：
--
作者：
Moeller NH;Shi K;Demir Ö;Belica C;Banerjee S;Yin L;Durfee C;Amaro RE;Aihara H
SARS-CoV-2非结构蛋白14(NSP14)外显子在病毒RNA合成和逃避宿主免疫反应过程中起着重要的校对作用。我们利用X射线结晶学、分子动力学模拟和生化分析研究了nsp14-外显子的结构、动力学和RNA结合机制，以及另一种病毒蛋白nsp10是如何调节其活性的。我们还证明了nsp14-外显子可以与病毒RNA聚合酶合作，在链终止药物存在的情况下使RNA合成，以生物化学的方式重现校对过程。我们的研究为一种关键的病毒酶的功能提供了机械性的见解，并为未来化学抑制剂的开发提供了基础。冠状病毒(CoV)大RNA基因组的高保真复制是由非结构蛋白14(NSP14)中的3‘到5’外显子(Exon)介导的，它剔除了包括抗病毒药物在内的核苷酸，这些核苷酸被低保真的病毒RNA依赖RNA聚合酶(RdRp)错误结合，并与病毒RNA重组和天然免疫耐受有关。在这里，我们确定了严重急性呼吸综合征冠状病毒2(SARS-CoV-2)外显子与其基本辅因子nsp10的复合体的1.6°分辨率的晶体结构。该结构显示出活性中心两侧的高度碱性和凹面，由nsp14的几个Lys残基和nsp10的N-末端氨基组成。建模表明，这个基本补丁与双链RNA底物的模板链结合，将新生的链的3‘端定位在外显子活性部位，这一点得到了突变和计算分析的证实。我们还表明，外显子的活性可以挽救被索索布韦中毒的停滞的RNA引物，并允许RdRp在链终止药物的存在下继续其延伸，生化重现SARS-CoV-2复制中的校对。分子动力学模拟进一步显示了多结构域nsp14的显著灵活性，并表明nsp10稳定了底物RNA结合的外显子，以支持其核酸外切酶活性。我们的SARS-CoV-2外显子-nsp10复合体的高分辨率结构为未来抗冠状病毒药物或减弱病毒毒力的策略提供了一个平台。
SARS-CoV-2 nonstructural protein 14 (nsp14) exoribonuclease (ExoN) plays important roles in the proofreading during viral RNA synthesis and the evasion of host immune responses. We used X-ray crystallography, molecular dynamics simulations, and biochemical assays to investigate the structure, dynamics, and RNA-binding mechanisms of nsp14-ExoN and how its activity is regulated by another viral protein, nsp10. We also demonstrated that nsp14-ExoN can collaborate with the viral RNA polymerase to enable RNA synthesis in the presence of a chain-terminating drug, biochemically recapitulating the proofreading process. Our studies provide mechanistic insights into the functions of a key viral enzyme and a basis for future development of chemical inhibitors. High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3′-to-5′ exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3′ end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN–nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.
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影响因子：
5
作者：
Gorbalenya AE;Enjuanes L;Ziebuhr J;Snijder EJ
通讯作者：
Snijder EJ
影响因子：
4.6
作者：
Jockusch S;Tao C;Li X;Chien M;Kumar S;Morozova I;Kalachikov S;Russo JJ;Ju J
通讯作者：
Ju J
DOI：
10.1073/pnas.96.24.13910
发表时间：
1999-11-23
影响因子：
11.1
作者：
Drake, JW;Holland, JJ
通讯作者：
Holland, JJ
影响因子：
3.9
作者：
Jenkins, GM;Rambaut, A;Holmes, EC
通讯作者：
Holmes, EC
DOI：
10.1074/jbc.m112.420521
发表时间：
2013-06-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Jiang X;Huang Q;Wang W;Dong H;Ly H;Liang Y;Dong C
通讯作者：
Dong C