Defining the Translocation Mechanisms of SARS-CoV-2 nsp13 Helicase to Aid in Antiviral Development

项目摘要 新冠肺炎的病原体SARS-CoV-2已在全球感染超过1.03亿人(2月 2021年)，死亡人数超过225万人，对美国人的健康和经济福祉构成了可怕的威胁 整个世界。尽管疫苗似乎对SARS-CoV-2有效，但最近关于 潜在的疫苗耐药株突显了抗击这种病毒的替代战略的重要性。这个 针对重要的抗突变病毒蛋白如nsp13的抗病毒治疗的发展就是其中之一。 策略。提高对nsp13利用的分子机制的了解对于合理地 开发抑制剂。该项目将利用集成的多尺度模型蛋白质来解决这一缺陷 结晶学和生化方法确定SARS-CoV-2 nsp13解旋酶如何与RNA和ATP结合 底物，在ATP结合和水解过程中传递能量，并在配基过程中改变构象 结合和催化。我们提出了以下建议：1)鉴定RNA的分子水平成分-- Nsp13的结合和转位机制。初步全原子分子动力学(AAMD)模拟 SARS-CoV-2的NSP13已经确定了关键的蛋白质-RNA相互作用，这些相互作用将为最初的突变研究提供信息。 进一步的模拟和蛋白质结晶学将有助于了解依赖于ATP的蛋白质-RNA相互作用 在RNA裂隙中观察到。将进行生化实验以测试结构-功能 基于结构的方法产生的假设。2)分子水平特征的鉴定 Nsp13对三磷酸腺苷的结合、水解和产物释放。我们已经对SARS进行了AAMD模拟- CoV-2 nsp13在所有相关底物状态。浸泡的三磷酸腺苷和非水解性类似蛋白 将进行结晶学测试这些初始模型。随后的量子力学计算 将确定ATP水解反应的关键成分。定点突变和成熟的 酶动力学分析将被用来检验这些模拟预测的效果。3)变构化合物的鉴定 SARS-CoV-2 nsp13中的网络，从ATP结合和水解中转换能量来执行RNA 易位。利用AAMD模拟的网络分析，Motif V被确定为关键的变构 贡献者。将进行生化研究，以证实基序V是nsp13解旋酶所必需的 功能。将做进一步的工作来确定ATP的其他组分之间的变构网络 在AIMS 2和3中发现了Pocket和RNA裂解。这项工作将产生前所未有的分子水平的洞察力 探讨SARS-CoV-2 nsp13解旋酶的易位机制。这一机制的关键部件 代表了抗病毒开发的新目标。

Project Summary SARS-CoV-2, the causative agent of COVID-19, has infected more than 103M people worldwide (February 2021) with more than 2.25M deaths, and represents a dire threat to the health and economic well-being of the entire world. Although vaccines seem to be effective against SARS-CoV-2, recent information regarding potential vaccine resistant strains highlights the importance of alternative strategies to combat this virus. The development of antiviral therapeutics on important mutation resistant viral proteins such as nsp13 is one such strategy. Improved knowledge of the molecular mechanisms utilized by nsp13 are necessary to rationally develop inhibitors. This project will address this deficiency utilizing an integrated multiscale modeling, protein crystallography, and biochemical approach to define how SARS-CoV-2 nsp13 helicase binds RNA and ATP substrates, transduces energy during ATP binding and hydrolysis, and changes conformation during ligand binding and catalysis. We propose the following: 1) Identification of molecular-level components of the RNA- binding and translocation mechanisms of nsp13. Preliminary all-atom molecular dynamics (aaMD) simulations of SARS-CoV-2 nsp13 have identified key protein-RNA interactions that will inform initial mutagenesis studies. Further simulation and protein crystallography will inform on the ATP-dependent protein-RNA interactions observed in the RNA cleft. Biochemical experiments will be performed to test the structure-function hypotheses generated by the structural-based approaches. 2) Identification of molecular-level features of the binding, hydrolysis and product release of ATP by nsp13. We have performed aaMD simulations of the SARS- CoV-2 nsp13 in all relevant substrate states. Soaked-in ATP and non-hydrolysable analogue protein crystallography will be performed to test these initial models. Subsequent quantum mechanical calculations will identify key components of the ATP hydrolysis reaction. Site-directed mutagenesis and well-established enzyme kinetics assays will be used to test effects predicted by these simulations. 3) Identification of allosteric networks in SARS-CoV-2 nsp13 that transduce energy from ATP binding and hydrolysis to perform RNA translocation. Utilizing network analyses of aaMD simulations, Motif V has been identified as a key allosteric contributor. Biochemical studies will be performed to verify that Motif V is necessary for nsp13 helicase function. Further work will be done to identify allosteric networks between additional components of the ATP pocket and RNA cleft identified in Aims 2 and 3. This work will produce unprecedented molecular-level insight into the translocation mechanism of SARS-CoV-2 nsp13 helicases. Key components of this mechanism represent new targets for antiviral development.