Catalytic Dyad Residues His41 and Cys145 Impact the Catalytic Activity and Overall Conformational Fold of the Main SARS-CoV-2 Protease 3-Chymotrypsin-Like Protease

Abstract

Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145–His41 catalytic dyad. We here characterized the catalytic roles of the cysteine–histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine–histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.

Phytochemicals-based targeting RdRp and main protease of SARS-CoV-2 using docking and steered molecular dynamic simulation: A promising therapeutic approach for Tackling COVID-19

“…From the binding frequency data, active site information for Mpro-ligand complexes over the simulation time showed a similar catalytic dyad of His41 and Cys145 [ 52 ], which has been reported previously [ [53] , [54] , [55] ]. Furthermore, the active site observations for RdRp were supported by other researchers such as Aftab et al, who report that ASP760 and ASP761 can be potential binding sites [ 56 , 57 ].…”

Discovery of 4′-O-methylscutellarein as a potent SARS-CoV-2 main protease inhibitor

“…3 , 4′- O -methylscutellarein ( 4 ) fitted well into the substrate binding site of M pro enzyme with the calculated binding energy of −17.1544 kJ/mol. Previous studies indicated that SARS-CoV-2 M pro catalyzes the cleavage of the protein substrate using the catalytic dyad His41 and Cys145 [ 31 ]. The docking result indicated that the 4′- O -methylscutellarein ( 4 ) is perfectly fitted into the core of the substrate-binding pocket and the catalytic dyad is sequestered from the solvent exposed area to prevent the peptide substrate approaching the active site ( Fig.…”

Aurasperone A Inhibits SARS CoV-2 In Vitro: An Integrated In Vitro and In Silico Study

“…Aurasperone A ( 3 ) showed high docking scores (−8.1, −8.0, and −7.8 kcal/mol, Table 1 ) towards the main protease (M pro ), helicase, and RdRp respectively. Visualization of the best docking pose of aurasperone A ( 3 ) against M pro showed the formation of H-bond with Cys-145 amino acid residue ( Figure 3 A and Figure S9A ), which is essential for the enzyme’s catalytic protease activity [ 29 ], in addition to Gly 143 and Ser-144 amino acids present in the active site of the enzyme. Aurasperone A (3) interacted with the ADP site of SARS-CoV-2 RNA helicase through hydrogen bonds with Arg-443 and Glu-540 residues and hydrophobic interaction with several other amino acid residue, as shown in Figure S9C [ 30 ].…”