{"doi":"10.1016/j.jbc.2025.110890","title":"A split luciferase system for studying coronavirus Mpro dimerization in vitro and in living cells","abstract":"The main protease enzyme (M pro ) of coronaviruses cleaves the viral polyprotein into functional units essential for virus replication.Prior work has demonstrated that M pro functions as a homodimer.However, studies on the mechanism of dimerization have been challenging because the purified protease is mostly dimeric, dimerization-defective mutants lack proteolytic activity, and robust cell-based assays have yet to be reported.To enable work on M pro dimerization, we have developed a quantitative luciferase-based SARS-CoV-2 (SARS2) M pro biosensor that accurately reports protein dimerization in living cells and, upon purification, also in vitro.Cotransfection of cells with a construct expressing M pro fused to the 18 kDa LargeBiT of luciferase (LgBiT) and a second construct with M pro fused to the 1 kDa SmallBiT of luciferase (SmBiT) results in a reconstitution of luciferase activity in a dose-dependent manner that requires conserved residues within the dimerization interface.Proteolytic activity is dispensable for dimerization and, uniquely, a C145A catalytically inactive mutant exhibits enhanced dimerization signal likely due to lower cytotoxicity.M pro enzymes from multiple different coronaviruses also dimerize in this system, indicating mechanistic conservation.Interestingly, this dimerization biosensor also provides a quantitative read-out of inhibitor-facilitated dimerization.Covalent SARS2 M pro inhibitors such as nirmatrelvir cause a 3-to 5-fold increase in luciferase activity.Together with corroborating structural, biophysical, and molecular dynamics experiments, our studies support a model in which covalent M pro inhibitors such as nirmatrelvir simultaneously block catalytic activity and induce allosteric stabilization of the dimeric complex.","journal":"Journal of Biological Chemistry","year":2025,"id":579983,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9596,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1490444,"name":"Jyoti Vishwakarma","orcid":"0000-0002-6536-2980","position":1,"is_corresponding":false},{"id":883805,"name":"Javier Sanlley-Hernandez","orcid":"0000-0002-7032-0996","position":2,"is_corresponding":false},{"id":1280793,"name":"Megan Tansiongco","orcid":"0009-0007-1375-7722","position":3,"is_corresponding":false},{"id":1281244,"name":"Ashley Cuell","orcid":null,"position":4,"is_corresponding":false},{"id":1280795,"name":"Agnieszka Dąbrowska","orcid":"0000-0003-1499-4950","position":5,"is_corresponding":false},{"id":731539,"name":"Rahul Basu","orcid":"0009-0005-7048-4017","position":6,"is_corresponding":false},{"id":368429,"name":"Philipp A. M. Schmidpeter","orcid":"0000-0003-2871-9706","position":7,"is_corresponding":false},{"id":1490445,"name":"You Hu","orcid":"0000-0002-2909-0594","position":8,"is_corresponding":false},{"id":307688,"name":"Susan E. Tsutakawa","orcid":"0000-0002-4918-4571","position":9,"is_corresponding":false},{"id":249874,"name":"Christina B. Cooley","orcid":"0000-0001-9699-7682","position":10,"is_corresponding":false},{"id":71085,"name":"Rommie E. Amaro","orcid":"0000-0002-9275-9553","position":11,"is_corresponding":false},{"id":257763,"name":"Reuben S. Harris","orcid":"0000-0002-9034-9112","position":12,"is_corresponding":false},{"id":1280791,"name":"Renee Delgado","orcid":"0000-0002-7148-4917","position":0,"is_corresponding":true}],"reference_count":60,"raw_metadata":null,"created_at":"2026-07-19T02:58:34.718602Z","pmid":"41197737","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}