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

Abstract

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.

Figure 1

A new GST fusion protein expression system. (a) The map of the pGSTM vector design. The cleavage site for SARS-CoV M^pro is labeled. (b) Expression and purification of calbindin D28k using the pGSTM expression system. Lane 1, total cell extract for calbindin D28k before induction; lane 2, total cell extract for calbindin D28k after induction overnight; lane 3, supernatant of the cell lysate; lanes 4 and 5, purified calbindin D28k; lane 6, protein molecular mass marker. (c) Schematic plot of WT SARS-CoV M^pro construct designed. (d) Expression and purification of WT M^pro . Lane 1, protein molecular mass marker; lane 2, total cell extract for WT M^pro before induction; lane 3, total cell extract for WT M^pro after induction overnight; lane 4, WT-GPH₆ after affinity chromatography; lane 5, WT after cleavage by rhinovirus 3C protease; lane 6, GPLGS-WT; lane 7, GS-WT.

Figure 2

Superposition of WT and GPLGS-WT M^pros. The substrate-binding pocket of one promoter is in surface representation. GPLGS-WT is in blue, WT is in magenta.

Figure 3

Superposition of the S1 pockets of GPLGS-WT and WT SARS-CoV M^pro (in stereo). (a) Superposition of the S1 pockets in protomer A of GPLGS-WT and that of protomer A* of WT SARS-CoV M^pro. Protomer A* of WT is in blue; protomer A of GPLGS-WT is in yellow; protomer B* of WT is in magenta; protomer B of GPLGS-WT is in red. In the WT structure, the amino group (NH₂) of Ser1 in protomer B* donates a 3.0 Å hydrogen bond to the carboxylate group of Glu166 and a 2.7 Å hydrogen bond to the main-chain carbonyl group of Phe140 in protomer A*, stabilizing the S1 pocket. The NH of Gly143 moves 0.8 Å toward the activity site; the main chain of residues 142-143 moves toward the S1 subsite; the side-chain of Asn-A*142 flips over with a 6 Å shift compared with protomer A of GPLGS-WT. (b) Superposition of the S1 pockets in protomer B of GPLGS-WT and that of Protomer A* of WT SARS-CoV M^pro. Protomer A* of WT is in blue; protomer B of GPLGS-WT is in yellow; protomer B* of WT is in magenta; protomer A of GPLGS-WT is in red. The S1 pocket of protomer B collapses partly with reorientation of Glu166 and residues 140–143. No electron density was visible for residues A1 and A2.

Figure 4

Differences between the complex structures of WT and GPLGS-WT. (a) Inhibitor N3. (b) Superposition of the substrate-binding pockets in protomer A of GPLGS-WT and that in protomer A* of WT. In the WT-N3 complex structure, the NH₂ group of Ser1 in protomer B* was still hydrogen-bonded to the carboxylate group of Glu166 and the carbonyl group of Phe140 in protomer A*, stabilizing the S1 pocket. In the GPLGS-WT-N3 complex structure, however, the two hydrogen bonds described above were not found. Instead, an ordered water molecule was observed in the S1 pocket. Protomer A* of WT is in blue; protomer A of GPLGS-WT is in yellow; inhibitor N3 (complexed with WT) is in magenta; inhibitor N3 (complexed with GPLGS-WT) is in red; protomer B* of WT is in green; protomer B of GPLGS-WT is in cyan.

Supplementary Data Figure S1

Supplementary Data Figure S2