1. Mutational profiling of SARS-CoV-2 papain-like protease reveals requirements for function, structure, and drug escape.
- Author
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Wu X, Go M, Nguyen JV, Kuchel NW, Lu BGC, Zeglinski K, Lowes KN, Calleja DJ, Mitchell JP, Lessene G, Komander D, Call ME, and Call MJ
- Subjects
- Humans, Coronavirus Papain-Like Proteases genetics, Coronavirus Papain-Like Proteases metabolism, Coronavirus Papain-Like Proteases chemistry, Catalytic Domain, Antiviral Agents pharmacology, Coronavirus 3C Proteases genetics, Coronavirus 3C Proteases metabolism, Coronavirus 3C Proteases antagonists & inhibitors, Coronavirus 3C Proteases chemistry, COVID-19 virology, COVID-19 Drug Treatment, Models, Molecular, HEK293 Cells, SARS-CoV-2 genetics, Mutation
- Abstract
Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process., (© 2024. The Author(s).)
- Published
- 2024
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