State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China.
FASEB J. 2019 Dec;33(12):14575-14587. doi: 10.1096/fj.201901624RR. Epub 2019 Nov 5.
Coronaviruses (CoVs) infect humans and multiple other animal species, causing highly prevalent and severe diseases. 3C-like proteases (3CLs) 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 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. 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, 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 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 in CoVs and other viral 3C proteases.-Zhou, J., Fang, L., Yang, Z., Xu, S., Lv, M., Sun, Z., Chen, J., Wang, D., Gao, J., Xiao, S. Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach.
冠状病毒（CoV）感染人类和多种其他动物物种，导致高度流行和严重的疾病。CoV 的 3C 样蛋白酶（3CLs，也称为主要蛋白酶）对于病毒复制至关重要，并且还参与多蛋白切割和免疫调节，使其成为开发抗病毒药物的有吸引力和有效的靶标。在此，我们以猪流行性腹泻病毒（一种肠致病性 CoV）的 3CL 作为模型，鉴定新型关键残基对酶活性的影响。首先，我们建立了一种快速、灵敏、高效的基于荧光素酶的生物传感器来监测 PDEV 3CL 的活性。使用这种荧光素酶生物传感器，以及确认了众所周知的催化残基（His41 和 Cys144），我们鉴定了 PDEV 3CL 的 4 种新型蛋白水解失活突变体，通过生化实验也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，发生在蛋白酶活性位点内部和外部的氢键相互作用以及底物的动态波动，特别是范德华接触，都发生了剧烈的改变，这种情况与 3CL 活性的丧失有关。这些数据表明，改变蛋白质-底物复合物的分子间动力学会消除蛋白酶活性的机制。在结合口袋中发现新型关键残基对酶活性的影响，可能为设计蛋白酶抑制剂提供更多可成药的靶标。此外，我们使用 MD 模拟对动态底物包络模型进行深入研究，这是一种可以增强针对 CoV 和其他病毒 3C 蛋白酶的新型抑制剂发现的方法。-Zhou, J., Fang, L., Yang, Z., Xu, S., Lv, M., Sun, Z., Chen, J., Wang, D., Gao, J., Xiao, S. 联合方法鉴定冠状病毒 3C 样蛋白酶中的新型蛋白水解失活突变。
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