Journal article
Mutational profiling of SARS-CoV-2 papain-like protease reveals requirements for function, structure, and drug escape
X Wu, M Go, JV Nguyen, NW Kuchel, BGC Lu, K Zeglinski, KN Lowes, DJ Calleja, JP Mitchell, G Lessene, D Komander, ME Call, MJ Call
Nature Communications | Published : 2024
Open access
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 fo..
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Awarded by Australian War Memorial
Funding Acknowledgements
This work has been supported by: a Wellcome Trust Innovator Award 222698/Z/21/Z to D.K., G.L., and M.J.C., WEHI Innovator funding to D.K., G.L., and M.J.C., MRFF grants MRF2002119 to D.K. and G.L., and MRF2016781 to D.K., G.L., and M.J.C.; NHMRC Investigator Grants (GNT2016461 to GL, GNT1178122 to DK); and a donation from John and Tibby Peterson to M.J.C. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector. The D.M.S. libraries were created with the help of the WEHI Multiplexed Assay Technology Hub, which was founded with WEHI New Medicines and Advanced Technology Theme funding. We would like to thank the Bio21-WEHI Crystallisation Facility within Melbourne Protein Characterisation at The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne. We thank WEHI Flow Cytometry and Advanced Genomics Facilities as well as the National Drug Discovery Centre for technical resources and advice. Work in the laboratories of the authors was made possible through Victorian State Government Operational Infrastructure Support (OIS); Australian Government NHMRC Independent Research Institute Infrastructure Support (IRIIS) Scheme; a donation from Hengyi Pacific Pty Ltd to support COVID-19 research; and a donation from AWM Electrical to support Australian drug discovery research to the National Drug Discovery Centre.