A split luciferase system for studying coronavirus Mpro dimerization in vitro and in living cells

The main protease enzyme (Mpro) of coronaviruses cleaves the viral polyprotein into functional units essential for virus replication. Prior work has demonstrated that Mpro functions as a homodimer. However, studies on the mechanism of dimerization have been challenging because the purified protease...

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Vydané v:The Journal of biological chemistry s. 110890
Hlavní autori: Delgado, Renee, Vishwakarma, Jyoti, Sanlley Hernandez, Javier O., Tansiongco, Megan, Cuell, Ashley, Dabrowska, Agnieszka, Basu, Rahul, Schmidpeter, Philipp A.M., Hu, You, Tsutakawa, Susan E., Cooley, Christina B., Amaro, Rommie E., Harris, Reuben S.
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: Elsevier Inc 01.11.2025
Predmet:
coronavirus protease
protease
protease dimerization
protease inhibitor
SARS-CoV-2 main protease (Mpro/3CLpro)
split-luciferase biosensor system
virus replication
protease dimerization
protease inhibitor
split-luciferase biosensor system
coronavirus protease
Mpro
virus replication
RMSF
SARS-CoV-2 main protease (Mpro/3CLpro)
SAXS
protease
SEC
SARS-CoV-2
SEC-MALS
MD
ITC
PLpro
ISSN:0021-9258, 1083-351X, 1083-351X
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Shrnutí:The main protease enzyme (Mpro) of coronaviruses cleaves the viral polyprotein into functional units essential for virus replication. Prior work has demonstrated that Mpro 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 Mpro dimerization, we have developed a quantitative luciferase-based SARS-CoV-2 (SARS2) Mpro biosensor that accurately reports protein dimerization in living cells and, upon purification, also in vitro. Co-transfection of cells with a construct expressing Mpro fused to the 18 kDa LargeBiT of luciferase (LgBiT) and a second construct with Mpro 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. Mpro 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 Mpro 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 Mpro inhibitors such as nirmatrelvir simultaneously block catalytic activity and induce allosteric stabilization of the dimeric complex.
Bibliografia:ObjectType-Article-1
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content type line 23
ISSN:0021-9258
1083-351X
1083-351X
DOI:10.1016/j.jbc.2025.110890