Correlation between dissociation and catalysis of SARS-CoV main protease

Abstract

The dimeric interface of severe acute respiratory syndrome coronavirus main protease is a potential target for the anti-SARS drug development. We have generated C-terminal truncated mutants by serial truncations. The quaternary structure of the enzyme was analyzed using both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. Global analysis of the combined results showed that truncation of C-terminus from 306 to 300 had no appreciable effect on the quaternary structure, and the enzyme remained catalytically active. However, further deletion of Gln-299 or Arg-298 drastically decreased the enzyme activity to 1-2% of wild type (WT), and the major form was a monomeric one. Detailed analysis of the point mutants of these two amino acid residues and their nearby hydrogen bond partner Ser-123 and Ser-139 revealed a strong correlation between the enzyme activity loss and dimer dissociation.

Two adjacent mutations on the dimer interface of SARS coronavirus 3C-like protease cause different conformational changes in crystal structure

“…Thus, the mutation might have substantially destabilized the dimer interface. This again demonstrates that the integrity of the dimer interface is essential for dimerization, as supported by a lot of reports that a single mutation on the dimer interface could cause the complete dissociation of the dimer (Chen et al, 2008a;Hsu et al, 2005b;Lin et al, 2008;Shi et al, 2008;Shi and Song, 2006).…”

“…The reliability of this hypothesis could be supported by the findings that mutations in the key interactions involved in this dimerization process could severely impair the dimer stability of SARS-CoV 3CL pro (Chen et al, 2008a;Hsu et al, 2005b;Lin et al, 2008;Shi et al, 2008;Shi and Song, 2006). These interactions include: (i) the initial association of the two domains III; (ii) the Hbonds between the two helices A′; (iii) the intra-protomer tethering between domain III and the N-finger; and (iv) the contact of domain III and the N-finger with the opposite S1 subsite.…”

“…SARS-CoV 3CL pro exists in a equilibrium of dimer and monomer in solution (Chou et al, 2004;Fan et al, 2004;Graziano et al, 2006aGraziano et al, , 2006bHsu et al, 2005aHsu et al, , 2005b, with a dissociation constant K D lower than 100 nM (Hsu et al, 2005b;Kuo et al, 2004;Verschueren et al, 2008). Since it has been widely proved that only the dimer is active (Barrila et al, 2006;Chang et al, 2007;Chen et al, 2005Chen et al, , 2006Chen et al, , 2008bChou et al, 2004;Fan et al, 2004;Graziano et al, 2006b;Hsu et al, 2005b;Lin et al, 2008), the dimer interface is regarded as an ideal target for structure-based enzyme inhibitor design. The major components of the interface are: (i) the N-terminal residues 1-7 (Nfinger), (ii) the S1 substrate-binding subsite, (iii) helix A′ (residues 10-15) that immediately follows the N-finger, and (iv) domain III.…”

Applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses

“…Mpro is a dimeric protein whose monomer has no catalytic activity [15,22,23]. Mpro is a dimeric protein whose monomer has no catalytic activity [15,22,23].…”

Lopinavir; A Potent Drug against Coronavirus Infection: Insight from Molecular Docking Study

“…The genome of SARS encodes 2 polyproteins namely ppla and pplb, with molecular weights of 450 and 750 KD, respectively. These polyproteins are cleaved to different functional proteins of spike, membrane, envelop, nucleoprotein, replicase, and polymerase (9)(10)(11). This process is performed by a chymotrypsin-fold proteinase of 33KD molecular mass.…”