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

Juliana C. Ferreira, Samar Fadl, Wael M. Rabeh

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number102023
JournalJournal of Biological Chemistry
Issue number6
StatePublished - Jun 2022


  • 3-chymotrypsin-like protease
  • 3CLpro
  • COVID-19
  • SARS-CoV-2
  • kinetic characterization
  • thermodynamic stability

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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