key: cord-0743152-36hzrd0l
authors: Ferreira, Juliana C.; Fadl, Samar; Rabeh, Wael M.
title: Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2
date: 2022-05-11
journal: J Biol Chem
DOI: 10.1016/j.jbc.2022.102023
sha: 55a59c95276dc484f7361525fafb4b0f3fc3e0c5
doc_id: 743152
cord_uid: 36hzrd0l

3C-like protease (3CLpro) is one of two proteases that process and liberate functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-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 2-fold 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 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 SARS-CoV-2 3CLpro.

in a one-turn -helix at the end of the N-finger. The side chains of S10 from both monomers form 4 .0Å H-bonding interactions, while the side chain of E14 forms a 2.8Å tight H-bond with the amide nitrogen of G11 and long-distance interactions with the side chain of K12 of each monomer at 4.5Å and 7.0Å. Third, the side chain of R4 of the N-finger of one monomer forms a salt bridge at 3.4Å or 3.6Å with E290 of domain III of the other monomer, again creating two contact points between the two monomers (Fig. 1D) . These salt bridge interactions are located in the middle of the dimer interface and are accompanied by tight H-bonding interactions at 2.9Å and 3.1Å between the side chains of S139 and Q299 of domains II and III, respectively. S139 is also part of the oxyanion hole formed by hydrogen bond donors of the main chain amides of the S139-L141 loop in the S1 subsite, which stabilizes formation of the oxyanion transition state during catalysis (11, (25) (26) (27) (28) (29) . Based on this structural analysis, S1, R4, S10, K12, E14, S139, E166, E290, and Q299

were selected for mutagenesis to characterize their roles in the activity and dimerization of SARS-CoV-2 3CLpro.

To assess the impact of the selected dimer interface residues of SARS-CoV-2 3CLpro on dimerization and activity, single amino acid substitutions were introduced. The mutants were expressed in E. coli and purified as previously reported for wild-type (WT) recombinant 3CLpro (32) . The oligomeric state of each purified protein was determined by analytical size-exclusion chromatography (aSEC). A minimum enzyme concentration of 7 mg/mL in 20 mM Hepes buffer pH 7.0 was used for all mutants and WT enzyme at 4 C, and the retention volumes were compared to assess perturbation of the dimer-monomer equilibrium (Fig. 2) . Similar to the WT enzyme, a single early peak corresponding to the dimer (71.6 kDa) was observed for S1A ( Fig. 2A) , S10A/C/T (Fig. 2C) , E14D (Fig. 2D) , and Q299A (Fig. 2F) . The other mutants exhibited both an early peak and a late peak, indicating an equilibrium between the dimer and monomer (35. Table S1 ). For R4A/Q, a single peak was observed with a smaller retention volume than that for the WT enzyme (Fig. 2B ). This earlier peak indicates that R4A/Q adopts a different shape or an oligomerization state that is larger than the dimeric state of the WT enzyme. In addition, two peaks were observed for S139A, E290A/D, and Q299E; the retention volume of the second peak was J o u r n a l P r e -p r o o f Page 6 of 27 similar to that of the WT enzyme ( Fig. 2F−2H) . The R4A/Q, S139A, E290A/D, and Q299E mutants were the only variants with a peak with a smaller retention volume than the WT enzyme.

The effects of the dimer interface mutations on overall thermodynamic stability were assessed by differential scanning calorimetry (DSC). The DSC thermograms of WT and mutant 3CLpro (Fig.   3 ) were acquired in 20 mM Hepes pH 7.0, 150 mM NaCl, and 20% (v/v) DMSO, similar to the buffer subsequently used to assay catalytic activity. The temperature was ramped from 15 °C to 75 °C at a scan rate of 1 °C/min to acquire the thermal unfolding transitions. The melting temperature (Tm) was calculated at the apex of the melting peak, and the calorimetric enthalpy (ΔHcal) was determined from the area under the thermographic peak. The WT enzyme exhibited a single transition with an early shoulder peak (Fig. 3A) . For all mutants, the overall shape of the thermogram was similar to that of WT, with a single thermal transition ( Fig. 3A values of the other mutants were close to that of WT ( Fig. S1A and S1B). Compared with WT, the majority of the dimer interface mutants exhibited significantly higher amplitudes of the DSC thermographic transition (Fig. 3A−3F ). For R4Q, S10C, E14Q, E14S, E290Q, and Q299N, ΔHcal increased by 3-fold compared with WT (133 ± 4 kJ/mol), with an average value of ~350 kJ/mol ( Fig. S1C and S1D).

The proteolytic activity of the dimer interface mutants was measured in 20 mM Hepes pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 20% (v/v) DMSO at a fixed concentration of the fluorescent peptide substrate of 60 µM as described previously (32) (33) (34) . This substrate concentration is equal to the Km of the WT enzyme, and the addition of 20% DMSO maximizes activity by reducing aggregation of the peptide substrate. The WT and mutant enzyme concentrations were varied from 0.5 to 5.0 µM, and the relative rate of each mutant was calculated by comparing the slope of the straight line of the plot of activity as a function of enzyme concentration to that of the WT enzyme ( Fig. 4A−4D) . Interestingly, mutation to alanine of the residues at the first site of interest in the dimer interface, S1 and E166 ( Fig. 1A and 1B) , 2-fold enhancement or reduced proteolytic activity to 89% of the WT enzyme, respectively ( Fig. 4A and J o u r n a l P r e -p r o o f S2A). The relative activity of 3CLPro S1A was double that of WT, while E166A exhibited 89% relative activity.

At the second dimer interface site of interest (Fig. 1C) , introduction of S10A or E14A eliminated 3CLpro proteolytic activity (Fig. 4B ). The role of S10 was further investigated by introducing S10C or S10T. Like S10A, 3CLPro S10C was inactive; by contrast, the activity of 3CLPro S10T was 90% of the WT activity ( Fig. 4B and S2A ). The inactivation of 3CLpro by E14A was further explored by examining the bonding interactions of E14 with the side chain of K12 (Fig. 1C) . The introduction of K12A reduced the activity of 3CLpro by 40% compared with WT (Fig. S2A ).

Alternative amino acid substitutions at E14, i.e., E14D, E14Q, and E14S, did not recover the activity of 3CLpro (Fig. 4B ).

Mutation of residues at the third dimer interface site of interest ( Fig. 1D ) greatly reduced 3CLpro activity. In particular, introduction of alanine at R4 or E290, which form ionic pairs at two contact points between the monomers, reduced relative activity to 16% or eliminated activity, respectively ( Fig. 4C ). Among alternative amino acid substitutions, 3CLPro R4Q had a relative activity of only 6%. All amino acid substitutions of E290, including E290D, E290Q, and E290Y, resulted in a lack of detectable proteolytic activity. In the same dimer interface pocket, S139 forms H-bonding interactions with Q299 (Fig. 1D) , and 3CLpro S139A, Q299A and Q299N exhibited relative activities of 46%, 4%, and 6%, respectively (Fig. 4D ).

To compare the kinetic parameters of the active mutants of 3CLpro with the WT enzyme, initial velocity studies were performed at 30 °C in 20 mM Hepes pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 20% (v/v) DMSO. The S1A mutation increased kcat by ~150% compared with WT, whereas all other mutations decreased kcat (Fig. 5A ). The reductions of kcat ranged from 2-fold for K12A and S139A to 4-fold for R4A (Table S2) , whereas S10T and E166A had smaller impacts on the kcat of 3CLpro. All mutations increased Km of 3CLpro by less than 2-fold compared with WT (62 ± 6 M); 3CLpro R4A had the lowest affinity for the peptide substrate ( Fig. 5B and Table S2 ). Thus, the effects of the dimer interface mutants on kcat were more pronounced than their effects on Km. Overall, the catalytic efficiency (kcat/Km) of S1A was unchanged compared with WT, whereas all other mutants exhibited reductions of catalytic efficiency (Fig. 5C ). The R4A mutant exhibited the largest decrease in catalytic efficiency, 7-fold, whereas the S10T, K12A, S139A, and E166A mutants displayed reductions of 2-to 3-fold.

In the fight against COVID-19 and the spread of SARS-CoV-2, the discovery of antiviral drugs and the development of therapeutics are of great importance. A conserved step in the maturation of coronaviruses is the processing of the replicase polyproteins to produce new virus particles. The 3CLpro protease is responsible for replicase polyprotein processing and the release of functional proteins during virus infection, making it an attractive target for the development of antiviral therapeutics against COVID-19. In this study, we evaluated the importance of dimer interface residues for the catalytic activity of SARS-CoV-2 3CLpro to support the screening and design of antivirals. Dimerization is thought to be essential for the activity of SARS-CoV-2 3CLpro, likely by ensuring the correct conformation of the Gly138-Leu141 loop that forms the oxyanion hole (13, 35) . Small molecules that target the dimer interface rather than the active site will not act as competitive inhibitors of 3CLpro, ensuring that inhibition cannot be overcome by increasing the substrate concentration. Such inhibitors would make more effective antivirals than molecules that bind the active site. In this study, three sites in the dimer interface were identified as potentially important for the dimerization of 3CLpro ( Fig. 1 ) and were targeted for mutagenesis to evaluate their roles in protease dimerization and activity.

Site 1: S1 and E166 S1 and E166 are expected to form H-bonding and ionic interactions at two points of contact between the two monomers of SARS-CoV-2 3CLpro. Specifically, the side chain of E166 forms H-bonding interactions with the side chain of S1 and tight ionic interactions with the -amine of the N-terminus ( Fig. 1A and 1B) . Interestingly, introduction of S1A increased the relative activity and catalytic turnover (kcat) of 3CLpro compared with WT; in SARS-CoV 3CLpro, the same mutation reduced relative activity by 54% compared with WT (23). The dimerization and catalytic efficiency (kcat/Km) of S1A were similar to those of WT SARS-CoV-2 3CLpro; similar results were obtained previously for mutation of this residue in SARS-CoV 3CLpro (23) . These results suggest that the interactions of S1 are not essential for the dimerization or activity of 3CLpro. Moreover, J o u r n a l P r e -p r o o f the finding that S1A increased activity has important implications for the virtual screening and design of small molecules that inhibit the activity of 3CLpro by binding at the dimer interface.

Disrupting the interactions of S1 may be undesirable, as doing so may increase the activity of SARS-CoV-2.

In SARS-CoV 3CLpro, E166 is thought to act as a link between the substrate binding site and the dimer interface (36) , and introduction of E166A in this enzyme decreased kcat by 2-fold compared with WT and eliminated the substrate-induced dimerization of the R298A mutant. In MERS 3CLpro, which exhibits weak dimerization that is enhanced in the presence of substrate, introduction of an alanine at this residue (E169) reduced kcat by ~6-fold and resulted in a shift to the monomer form even in the presence of substrate (37) . In the present study, introduction of E166A retained 89% of the WT proteolytic activity and had little effect on the dimer:monomer equilibrium. In addition to their side chain interaction, Ser1 and Glu166 are in close vicinity to Phe140, His163, and His172, key residues involved in the binding of peptide-substrate (38) .

Molecular dynamic simulations on 3CLpro from both SARS-CoV-2/CoV confirmed that the protonation state of His172 and its interaction with Phe140, His163, and Glu166 are important in shaping the S1 substrate-binding pocket of 3CLpro (39, 40) . Virtual and X-ray-based screens have suggested a role of E166 in non-covalent interactions with potential inhibitors of SARS-CoV-2 3CLpro (30, 41) , but this study is the first to evaluate the impact of this residue on activity and dimerization. In summary, the results of the present study suggest that the interactions of S1 and E166, whether with each other, other residues or substrate, are not major contributors for the dimerization or activity of 3CLpro.

The second site of interest at the dimer interface comprises residues S10, K12, and E14, which are located in a one-turn -helix at the end of the N-finger; the two monomers interact with one another at this site to form a single contact point at the dimer interface (Fig. 1C ). Similar interactions have been proposed for SARS-CoV 3CLpro (42) . In SARS-CoV 3CLpro, introduction of S10A eliminated activity and reduced the share of the dimer form by ~20% (23) . In the present study, SARS-CoV-2 3CLpro S10A was also inactive, as was S10C, which is unable to facilitate Hbonding interactions between the two monomers. By contrast, S10T was fully active, despite the larger size of the threonine side chain compared with serine. Thus, the H-bonding interactions mediated by the side chain of S10 in both monomers, not the size of the side chain, are important for the catalytic activity of 3CLpro. Unlike SARS-CoV 3CLpro S10A, the S10 mutants of SARS-CoV-2 3CLpro were largely present as dimers.

The side chain of E14 forms long-range (~4.5Å and 7Å) ionic interactions with the side chain of K12 but strong H-bonding interactions with the backbone amine nitrogen of Gly11 (Fig. 1C) . The introduction of K12A reduced the relative activity of 3CLpro by ~40% (Fig. S2A) , indicating that the interaction between the side chains of K12 and E14 is important but not crucial for protease activity. However, 3CLpro E14A was catalytically inactive, consistent with an important role of the interaction of the side chain of E14 with the backbone of Gly11 for 3CLpro activity. Further support for this role is provided by the inactivity of 3CLpro E14Q, despite the similar molecular sizes of glutamine and glutamate. Moreover, 3CLpro E14D and E14S were also inactive, suggesting that the distance between the N-fingers of the monomers and the polarity of the side chain of E14 are important for catalytic activity. Previous mutagenesis studies have examined only E14A in SARS-CoV 3CLpro; this mutant exhibited a decrease in the dimer form of ~50% and 4% relative activity compared with WT (23).

The analysis of the oligomerization of these mutants emphasized that dimerization of 3CLpro is not sufficient for protease activity, even when the active site is intact (Fig. S2A−S2B) . Like WT, the active mutant S10T and the inactive mutants S10A/C and E14A/D/Q/S were largely present as dimers. By contrast, the monomer form was more dominant at 60% for the reduced activity mutant K12A (Table S1) , and the inactive mutant E14A also exhibited a shoulder on aSEC corresponding to the monomer form. The reduced activity of 3CLpro K12A compared with the WT enzyme may be attributable to this increase in the monomer form. Regardless of activity or oligomeric state, all mutations in the N-finger of 3CLpro increased the ΔHcal of the protein as measured by DSC, with only small changes in Tm (Fig. S1A−S1D) . These increases in ΔHcal are attributable to changes in the overall bonding characteristics of the protein and indicate enhanced polar bonding interactions and hydrophilicity (43) (44) (45) . Overall, the analyses of mutations at this site suggest that the interactions initiated by S10 or E14 are critical for the activity of 3CLpro but not its dimerization.

J o u r n a l P r e -p r o o f Site 3: R4, S139, E290, and Q299

The third site of interest in the dimer interface includes the salt bridge between the side chains of R4 and E290 of the N-finger and domain III, respectively, and the H-bonding interactions between the side chains of S139 and Q299 of domains II and III, respectively (Fig. 1D) . The four residues of the third site create two contact points between the monomers that enhance the stability of the dimeric state. These residues are also conserved in SARS-CoV 3CLpro and participate in similar interactions (42, 46) .

An important role of R4 in coronavirus 3CLpro was first identified in mutagenesis studies targeting the N-and C-termini of SARS-CoV 3CLpro; removal of residues 1-3 from the N-terminus had no effect on dimerization and reduced relative activity by 24%, whereas removal of residues 1-4 largely eliminated both activity and dimerization (19). Similarly, Chen et al. (23) found that SARS-CoV 3CLpro R4A exhibited 10% relative activity and a 20% decrease in dimerization. However, Chou et al. (47) observed that mutation of R4 to alanine had very little effect on activity but decreased dimerization by 80%. In the present study, SARS-CoV-2 3CLpro R4A and R4Q exhibited 16% and 6% relative activity, respectively, with both mutants mostly dimeric with a smaller retention volume on aSEC than the WT enzyme (Fig. 2B) . Even though S1 and R4 are both part of the long loop at the N-terminus and interact with Domains II and III of the other monomer, respectively ( Fig. 1A and 1D) , mutation of these residues to alanine had very different effects on the catalytic activity of 3CLpro: S1A and R4A had relative activities of 200% and 16%, respectively ( Fig. S2A−S2B) .

The importance of R4 in maintaining 3CLpro activity is most likely due to its interactions with E290, as all mutations of the latter residue eliminated protease activity. Like the R4 mutants, the E290 mutants had diverse oligomeric states, with larger retention volumes for E290A/D and smaller retention volumes for E290Q/Y compared with the WT enzyme. Introduction of E290A was previously reported to eliminate activity for SARS-CoV 3CLpro (47) . Even though are present as dimers, the lack of catalytic capabilities of R4A/Q and E290A/D mutants can be a result of formation of a new type of dimer that has been shown previously for 3CLpro of SARS-CoV to be catalytically inactive (22) . The low proteolytic activity in the presence of R4A mutant was also J o u r n a l P r e -p r o o f observed in 3CLpro from Porcine epidemic diarrhea virus (PEDV), where dimeric state of the protease was maintained (16) .

In addition to its interactions with Glu290, Arg4 interacts at 2.8Å with the backbone carbonyl oxygen of Lys137 of the oxyanion L1 loop (residues 138-145) (17, 48) . These key interactions shape the S1 substrate-binding pocket in addition to His163 and Glu166 that assist in binding the peptide substrates, and Phe140 and His172 in maintaining the open-state of 3CLpro (17, 36, 48) .

MD simulation also revealed the importance of residues Gly143, Ser144, and Cys145 of the L1 loop in the inhibition of 3CLpro (49) . The L1 loop has been shown to be a promising target to improve the design of selective and efficient inhibitors targeting the 3CLpro of SARS-CoV-2 (49) .

Among the other residues in the R4/E290 pocket, Q299 was more important for the catalytic activity of SARS-CoV-2 3CLpro than S139; S139A exhibited 46% relative activity, whereas only residual activity of Q299A/N was observed (Fig. S2A−S2B) . Consistent with these findings, in a previous study, the Q299A and Q299N mutants of SARS-CoV 3CLpro exhibited reductions of kcat of ~45-fold and ~19-fold, respectively, while S139A exhibited no reduction of activity or dimerization compared with WT (46) . Molecular dynamics simulations of ligand complexes of SARS-CoV-2 3CLpro suggest that a water-mediated interaction between S139 of one monomer and Q299 of the other monomer links dimerization with appropriate active site conformation, as S139 is also part of the oxyanion hole (50) .

Like the portion of the N-finger (site 2) described above, no clear relationship between oligomeric state and activity was observed for mutations in this third site of the dimer interface of 3CLPro.

The residually active mutants Q299A/N and the inactive mutant Q299E were all present in dimer form, although the retention volumes of Q299E/N were larger than that of the WT enzyme ( Fig.   2F and S2B) . However, R4A and R4Q, which exhibited similarly low levels of residual activity, were present in primarily dimeric form (Fig. 3B) . Moreover, the inactive mutants E290A/D were present as dimers, but the similarly inactive mutants E290Q/Y exhibited a shift toward the monomer state. DSC revealed that among all mutations at all sites, the inactive mutant E290A had the highest Tm, 49.5 ± 0.2 C (Fig. S1B) .

The introduction of mutations of key residues of the dimer interface of SARS-CoV-2 3CLpro confirmed the importance of some of these residues for dimerization and activity. All of the 3CLpro mutants with at least 50% relative activity, with the exception of K12A, exhibited an oligomer state similar to that of the dimeric WT enzyme, including S1A, S10T, S139A and E166A.

The K12A mutant exhibited an equilibrium between the two states, with a preference for the monomer, and 60% relative activity compared with WT. By contrast, the oligomeric states of the inactive mutants spanned a wide range, with aSEC retention volumes that were similar, larger or smaller than that of the WT enzyme. The results presented here demonstrate that dimerization does not guarantee a catalytically active 3CLpro, where some mutants that formed dimers were inactive.

As has been shown previously for SARS-CoV 3CLpro (22) , the dimeric form of catalytically inactive mutants can be a result of formation of a new type of dimers that are different from that of the WT enzyme. There was also no clear link between thermodynamic stability and activity: S10T, which was as active as WT, had Tm and ΔHcal values similar to those of WT, whereas S1A, which had higher activity than the WT enzyme, exhibited a 2-fold increase in ΔHcal compared with WT (Fig. 3G ).

This study is one of the first to directly probe the roles of dimer interface residues of SARS-CoV-2 3CLpro through mutagenesis, biophysical characterization, and activity assays. A number of studies have performed virtual or high-throughput structural biology-based screens of potential inhibitors of 3CLpro (41, 51, 52) , but the findings have not been complemented by biochemical assessments of the roles of specific residues or the effects of proposed inhibitors. The computational screening of inhibitors that bind the active site of 3CLpro will more likely yield competitive inhibitors that are less potent than non-competitive inhibitors targeting allosteric sites in the dimer interface. The work described here will help direct computational screening efforts to develop effective inhibitors that bind the dimer interface.

with carbonic anhydrase (29.6 kDa) and ribokinase (70 kDa) as marker proteins mimicking the monomeric and dimeric forms of the WT enzyme, which have molecular weights of 34.5 and 69 kDa, respectively.

The thermodynamic stability of 3CLpro was measured using a Nano DSC instrument (TA Instruments) calibrated using chicken egg white lysozyme, a known external Nano DSC standard that is part of the TA Instruments test kit (602198.901). The thermogram was acquired at a 3CLpro concentration of 30 M in 20 mM Hepes pH 7.0, 150 mM NaCl, and 20% (v/v) DMSO. The protein samples were heated from 15 °C to 75 °C at a scan rate of 1 °C/min and 3 atm pressure.

The background scans were obtained by loading degassed buffer in both the reference and sample cells and heating at the same rate. The DSC thermograms were corrected by subtracting the corresponding buffer baseline and converted to plots of excess heat capacity (Cp) as a function of temperature. The Tm was determined at the maximum temperature of the thermal transition, and the ΔHcal of the transition was estimated from the area under the thermal transition using Nano Analyzer software (TA Instruments).

The catalytic activities of WT and mutant 3CLpro were measured by a fluorescence resonance energy transfer (FRET)-based assay using the 14-amino-acid fluorogenic peptide substrate (DABCYL)KTSAVLQ↓SGFRKME(EDANS)-NH2 (GenScript Inc.; Piscataway, NJ) as described previously (10, (53) (54) (55) (56) . Error bars were calculated from triplicate measurements of each reaction, and the results are presented as the mean ± standard deviation (SD). indicating dimer formation. The retention volume of Q299A/N was similar to that of WT. By contrast, a major peak with a smaller retention volume than WT was observed for S139A or (A-F) DSC thermograms of the dimer interface mutants of 3CLpro. All enzymes exhibited a single transition with an early shoulder peak. For all mutants, the thermogram amplitude was higher than that for WT. Data were confirmed by triplicate runs. Bar plots of kcat and Km values of WT and dimer interface mutants of 3CLpro. All mutants exhibited decreased kcat except S1A, which had higher kcat than WT. In addition, all mutants had reduced affinity for the peptide substrate, as indicated by increases in their Km values compared with WT. (C) Bar plot of the catalytic efficiency (kcat/Km) of the dimer interface mutants of 3CLpro. R4A had the lowest catalytic efficiency, whereas the catalytic efficiency of S1A was equivalent to that of WT. Thus, these residues have different impacts on the activity of 3CLpro

Evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as coronavirus disease 2019 (COVID-19) pandemic: A global health emergency

World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19)

Reveals Mosaic Pattern of Phylogeographical Distribution. mSystems 5

The proximal origin of SARS-CoV-2

SARS-CoV-2, SARS-CoV, and MERS-COV: A comparative overview

Origin and evolution of pathogenic coronaviruses

Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China

Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses

Current Status of Epidemiology, Diagnosis, Therapeutics, and Vaccines for Novel Coronavirus Disease 2019 (COVID-19)

Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs

The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain

Long-range cooperative interactions modulate dimerization in SARS 3CLpro

Mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3C-like protease

Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CL(pro)

Ligand-induced dimerization of middle east respiratory syndrome (MERS) coronavirus nsp5 protease (3CLpro): implications for nsp5 regulation and the development of antivirals

Structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3C-like protease

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors

3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism

The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase

Without its N-finger, the main protease of severe acute respiratory syndrome coronavirus can form a novel dimer through its C-terminal domain

Residues on the dimer interface of SARS coronavirus 3C-like protease: dimer stability characterization and enzyme catalytic activity analysis

Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme

Enzyme catalysis by hydrogen bonds: the balance between transition state binding and substrate binding in oxyanion holes

Computational design of catalytic dyads and oxyanion holes for ester hydrolysis

Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease

Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological Cterminal autoprocessing site

Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication

Biochemical and biophysical characterization of the main protease, 3-chymotrypsin-like protease (3CLpro) from the novel coronavirus SARS-CoV 2

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

Dimethyl sulfoxide reduces the stability but enhances catalytic activity of the main SARS-CoV-2 protease 3CLpro

Activation and maturation of SARS-CoV main protease

Mutation of Glu-166 blocks the substrateinduced dimerization of SARS coronavirus main protease

Critical Assessment of the Important Residues Involved in the Dimerization and Catalysis of MERS Coronavirus Main Protease

The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor

) pHdependent Conformational Flexibility of the SARS-CoV Main Proteinase (Mpro) Dimer: Molecular Dynamics Simulations and Multiple X-ray Structure Analyses

Quaternary Structure of the SARS Coronavirus Main Protease

A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study

The Influence of Processing Parameters on Food Protein Functionality I. Differential Scanning Calorimetry as an Indicator of Protein Denaturation

The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation

Correlation between dissociation and catalysis of SARS-CoV main protease

Quaternary structure of the severe acute respiratory syndrome (SARS) coronavirus main protease

Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain

Multiscale Simulations of SARS-CoV-2 3CL Protease Inhibition with Aldehyde Derivatives. Role of Protein and Inhibitor Conformational Changes in the Reaction Mechanism

SARS-COV-2 M(pro) conformational changes induced by covalently bound ligands

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*

Progress in Developing Inhibitors of SARS-CoV-2 3C-Like Protease

Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction

From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design

Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay

FRET-Based Assays to Determine Calpain Activity

The authors declare that they have no conflicts of interest with the contents of this article.