Mutational profiling of SARS-CoV-2 papain-like protease reveals requirements for function, structure, and drug escape

Xinyu Wu, Margareta Go, Julie V. Nguyen, Nathan W. Kuchel, Bernadine G. C. Lu, Kathleen Zeglinski, Kym N. Lowes, Dale J. Calleja, Jeffrey P. Mitchell, Guillaume Lessene, David Komander, Matthew E. Call and Melissa J. Call ()
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Xinyu Wu: The Walter and Eliza Hall Institute of Medical Research
Margareta Go: The Walter and Eliza Hall Institute of Medical Research
Julie V. Nguyen: The Walter and Eliza Hall Institute of Medical Research
Nathan W. Kuchel: The Walter and Eliza Hall Institute of Medical Research
Bernadine G. C. Lu: The Walter and Eliza Hall Institute of Medical Research
Kathleen Zeglinski: The Walter and Eliza Hall Institute of Medical Research
Kym N. Lowes: The Walter and Eliza Hall Institute of Medical Research
Dale J. Calleja: The Walter and Eliza Hall Institute of Medical Research
Jeffrey P. Mitchell: The Walter and Eliza Hall Institute of Medical Research
Guillaume Lessene: The Walter and Eliza Hall Institute of Medical Research
David Komander: The Walter and Eliza Hall Institute of Medical Research
Matthew E. Call: The Walter and Eliza Hall Institute of Medical Research
Melissa J. Call: The Walter and Eliza Hall Institute of Medical Research

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-50566-9

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