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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



Faseb Journal 33(12): 14575-14587



Coronaviruses (CoVs) infect humans and multiple other animal species, causing highly prevalent and severe diseases. 3C-like proteases (3CLpros) 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 3CLpro 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 3CLpro 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 3CLpro, 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 3CLpro 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 3CLpro 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.

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Accession: 069485702

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PMID: 31690127


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