Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach
Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach
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使用组合方法鉴定冠状病毒 3C 样蛋白酶中的新型蛋白水解无活性突变
DOI：
10.1096/fj.201901624rr
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发表时间：
2019-12-01
期刊：
影响因子：
4.8
通讯作者：
Xiao, Shaobo
中科院分区：
文献类型：
--
作者：
Zhou, Junwei;Fang, Liurong;Xiao, Shaobo
冠状病毒（CoV）感染人类和多种其他动物物种，引起高度流行和严重的疾病。来自CoV的3C样蛋白酶（3CL（pro）s）（也称为主要蛋白酶）对于病毒复制是必需的，并且还参与多蛋白切割和免疫调节，使它们成为开发抗病毒药物的有吸引力的和有效的靶标。本文中，来自猪流行性腹泻病毒（一种肠致病性CoV）的3CL（pro）被用作鉴定酶活性的新的关键残基的模型。首先，我们建立了一个快速，灵敏，高效的基于生物传感器的PDEV 3CL（pro）在体内的活动进行监测。利用该荧光素酶生物传感器，沿着对已知催化残基（His 41和Cys 144）的确认，我们鉴定了4种新的PDEV 3CL（pro）蛋白水解失活突变体，这也通过生物化学实验在哺乳动物细胞中得到了证实。我们的分子动力学（MD）模拟表明，氢键相互作用发生在内部和外部的蛋白酶的活性位点和动态波动的基板，特别是货车的德瓦尔斯接触，急剧改变，有关的损失的情况3 CL（亲）的活动。这些数据表明，改变蛋白质-底物复合物中的分子间动力学消除了蛋白酶活性的机制。结合口袋中酶活性的新的关键残基的发现可能为蛋白酶抑制剂的设计提供更多的药物位点。此外，我们使用MD模拟对动态底物的包膜模型进行的深入研究是一种可以增加在CoV和其他病毒3C蛋白酶中发现针对3CL（pro）的新抑制剂的方法。
Coronaviruses (CoVs) infect humans and multiple other animal species, causing highly prevalent and severe diseases. 3C-like proteases (3CL(pro)s) from CoVs (also called main proteases) are essential for viral replication and are also involved in polyprotein cleavage and immune regulation, making them attractive and effective targets for the development of antiviral drugs. Herein, the 3CL(pro) from the porcine epidemic diarrhea virus, an enteropathogenic CoV, was used as a model to identify novel crucial residues for enzyme activity. First, we established a rapid, sensitive, and efficient luciferase-based biosensor to monitor the activity of PDEV 3CL(pro) in vivo. Using this luciferase biosensor, along with confirming the well-known catalytic residues (His41 and Cys144), we identified 4 novel proteolytically inactivated mutants of PDEV 3CL(pro), which was also confirmed in mammalian cells by biochemical experiments. Our molecular dynamics (MD) simulations showed that the hydrogen bonding interactions occurring within and outside of the protease's active site and the dynamic fluctuations of the substrate, especially the van der Waals contacts, were drastically altered, a situation related to the loss of 3CL(pro) activity. These data suggest that changing the intermolecular dynamics in protein-substrate complexes eliminates the mechanism underlying the protease activity. The discovery of novel crucial residues for enzyme activity in the binding pocket could potentially provide more druggable sites for the design of protease inhibitors. In addition, our in-depth study of the dynamic substrate's envelope model using MD simulations is an approach that could augment the discovery of new inhibitors against 3CL(pro) in CoVs and other viral 3C proteases.