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

Abstract

The 3C‐like protease of the severe acute respiratory syndrome (SARS) coronavirus has a C‐terminal extra domain in addition to the chymotrypsin‐fold adopted by piconavirus 3C proteases hosting the complete catalytic machinery. Previously we identified the extra domain to be involved in enzyme dimerization which has been considered essential for the catalytic activity. In an initial attempt to map out the extra‐domain residues critical for dimerization, we have systematically generated 15 point mutations, five deletions and one triple mutation and subsequently characterized them by enzymatic assay, dynamic light scattering, CD and NMR spectroscopy. The results led to identification of four regions critical for enzyme dimerization. Interestingly, Asn214Ala mutant with a significant tendency to form a monomer still retained ≈ 30% activity, indicating that the relationship between the activity and dimerization might be very complex. Very surprisingly, two regions (one over Ser284–Thr285–Ile286 and another around Phe291) were discovered on which Ala‐mutations significantly increased the enzymatic activities. Based on this, a super‐active triple‐mutant STI/A with a 3.7‐fold activity enhancement was thus engineered by mutating residues Ser284, Thr285 and Ile286 to Ala. The dynamic light scattering, CD and NMR characterizations indicate that the wild‐type (WT) and STI/A mutant share similar structural and dimerization properties, thus implying that in addition to dimerization, the extra domain might have other mechanisms to regulate the catalytic machinery. We rationalized these results based on the enzyme structure and consequently observed an interesting picture: the majority of the dimerization‐critical residues plus Ser284–Thr285–Ile286 and Phe291 are clustered together to form a nano‐scale channel passing through the central region of the enzyme. We therefore speculate that this channel might play a role in relaying regulatory effects from the extra domain to the catalytic machinery.

Mutation of Gly-11 on the Dimer Interface Results in the Complete Crystallographic Dimer Dissociation of Severe Acute Respiratory Syndrome Coronavirus 3C-like Protease

“…Besides the N-terminal finger, it has been reported that domain III also contributes well to dimerization of SARS-CoV 3CL pro (6,14). In agreement with this result, domain III undergoes a dramatic conformational switch relative to the chymotrypsin fold (domain I and II) in G11A monomer, rotating by 24°comparing with its normal position in wild-type dimer.…”

“…Why Does Mutation of Gly-11 Cause the Complete Dissociation of the Dimer in Crystal?-To date, the N-terminal finger (residues 1-7) and domain III have been identified to extensively mediate monomer-monomer interactions of SARS-CoV 3CL pro (6,10,14,15,18). Now we have brought forward a new structural element that is also vital for dimerization of the protease, namely ␣-helix AЈ (residues 10 -15).…”

“…Furthermore, the residues of domain III were revealed to extensively mediate monomer-monomer interactions and be responsible for positioning the N-terminal finger of one monomer to interact with the active site of the other monomer (6,14). In addition, we observed that the ␣-helix AЈ of domain I (residues 10 -15) is also a critical part of the dimer interface and contributes well to the dimer stability and catalytic activity of SARS-CoV 3CL pro .…”

“…To date, the contributions of the residues on the dimer interface to SARS-CoV 3CL pro dimerization and enzymatic activity have been identified by several groups (10,14,15). The N-terminal finger of the protease is considered to play an important role in both dimerization and activity.…”

Correlation between dissociation and catalysis of SARS-CoV main protease

“…Each subunit contains a catalytic dyad defined by His41 and Cys145 in the active site (Fig. However, the enzyme is catalytically compe-tent only after dimer formation [9][10][11][12][13][14][15][16][17][18][19][20]. However, the enzyme is catalytically compe-tent only after dimer formation [9][10][11][12][13][14][15][16][17][18][19][20].…”