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Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2

3C-like protease (3CLpro) processes and liberates functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome coronavirus 2, the virus responsible for COVID-19. It has been suggested that 3CLpro is catalytically active as a dimer, making the dimerizati...

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Autores principales: Ferreira, Juliana C., Fadl, Samar, Rabeh, Wael M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9091064/
https://www.ncbi.nlm.nih.gov/pubmed/35568197
http://dx.doi.org/10.1016/j.jbc.2022.102023
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author Ferreira, Juliana C.
Fadl, Samar
Rabeh, Wael M.
author_facet Ferreira, Juliana C.
Fadl, Samar
Rabeh, Wael M.
author_sort Ferreira, Juliana C.
collection PubMed
description 3C-like protease (3CLpro) processes and liberates functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome coronavirus 2, the virus responsible for COVID-19. It has been suggested that 3CLpro is catalytically active as a dimer, making the dimerization interface a target for antiviral development. Guided by structural analysis, here we introduced single amino acid substitutions at nine residues at three key sites of the dimer interface to assess their impact on dimerization and activity. We show that at site 1, alanine substitution of S1 or E166 increased by twofold or reduced relative activity, respectively. At site 2, alanine substitution of S10 or E14 eliminated activity, whereas K12A exhibited ∼60% relative activity. At site 3, alanine substitution of R4, E290, or Q299 eliminated activity, whereas S139A exhibited 46% relative activity. We further found that the oligomerization states of the dimer interface mutants varied; the inactive mutants R4A, R4Q, S10A/C, E14A/D/Q/S, E290A, and Q299A/E were present as dimers, demonstrating that dimerization is not an indication of catalytically active 3CLpro. In addition, present mostly as monomers, K12A displayed residual activity, which could be attributed to the conspicuous amount of dimer present. Finally, differential scanning calorimetry did not reveal a direct relationship between the thermodynamic stability of mutants with oligomerization or catalytic activity. These results provide insights on two allosteric sites, R4/E290 and S10/E14, that may promote the design of antiviral compounds that target the dimer interface rather than the active site of severe acute respiratory syndrome coronavirus 2 3CLpro.
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spelling pubmed-90910642022-05-11 Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2 Ferreira, Juliana C. Fadl, Samar Rabeh, Wael M. J Biol Chem Research Article 3C-like protease (3CLpro) processes and liberates functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome coronavirus 2, the virus responsible for COVID-19. It has been suggested that 3CLpro is catalytically active as a dimer, making the dimerization interface a target for antiviral development. Guided by structural analysis, here we introduced single amino acid substitutions at nine residues at three key sites of the dimer interface to assess their impact on dimerization and activity. We show that at site 1, alanine substitution of S1 or E166 increased by twofold or reduced relative activity, respectively. At site 2, alanine substitution of S10 or E14 eliminated activity, whereas K12A exhibited ∼60% relative activity. At site 3, alanine substitution of R4, E290, or Q299 eliminated activity, whereas S139A exhibited 46% relative activity. We further found that the oligomerization states of the dimer interface mutants varied; the inactive mutants R4A, R4Q, S10A/C, E14A/D/Q/S, E290A, and Q299A/E were present as dimers, demonstrating that dimerization is not an indication of catalytically active 3CLpro. In addition, present mostly as monomers, K12A displayed residual activity, which could be attributed to the conspicuous amount of dimer present. Finally, differential scanning calorimetry did not reveal a direct relationship between the thermodynamic stability of mutants with oligomerization or catalytic activity. These results provide insights on two allosteric sites, R4/E290 and S10/E14, that may promote the design of antiviral compounds that target the dimer interface rather than the active site of severe acute respiratory syndrome coronavirus 2 3CLpro. American Society for Biochemistry and Molecular Biology 2022-05-11 /pmc/articles/PMC9091064/ /pubmed/35568197 http://dx.doi.org/10.1016/j.jbc.2022.102023 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Ferreira, Juliana C.
Fadl, Samar
Rabeh, Wael M.
Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2
title Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2
title_full Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2
title_fullStr Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2
title_full_unstemmed Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2
title_short Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2
title_sort key dimer interface residues impact the catalytic activity of 3clpro, the main protease of sars-cov-2
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9091064/
https://www.ncbi.nlm.nih.gov/pubmed/35568197
http://dx.doi.org/10.1016/j.jbc.2022.102023
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