Mutation of Glu-166 Blocks the Substrate-Induced Dimerization of SARS Coronavirus Main Protease

Abstract

The maturation of SARS coronavirus involves the autocleavage of polyproteins 1a and 1ab by the main protease (Mpro) and a papain-like protease; these represent attractive targets for the development of anti-SARS drugs. The functional unit of Mpro is a homodimer, and each subunit has a His-41cdots, three dots, centeredCys-145 catalytic dyad. Current thinking in this area is that Mpro dimerization is essential for catalysis, although the influence of the substrate binding on the dimer formation has never been explored. Here, we delineate the contributions of the peptide substrate to Mpro dimerization. Enzyme kinetic assays indicate that the monomeric mutant R298A/L exhibits lower activity but in a cooperative manner. Analytical ultracentrifugation analyses indicate that in the presence of substrates, the major species of R298A/L shows a significant size shift toward the dimeric form and the monomer-dimer dissociation constant of R298A/L decreases by 12- to 17-fold, approaching that for wild-type. Furthermore, this substrate-induced dimerization was found to be reversible after substrates were removed. Based on the crystal structures, a key residue, Glu-166, which is responsible for recognizing the Gln-P1 of the substrate and binding to Ser-1 of another protomer, will interact with Asn-142 and block the S1 subsite entrance in the monomer. Our studies indicate that mutation of Glu-166 in the R298A mutant indeed blocks the substrate-induced dimerization. This demonstrates that Glu-166 plays a pivotal role in connecting the substrate binding site with the dimer interface. We conclude that protein-ligand and protein-protein interactions are closely correlated in Mpro.

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

“…Based on the c(s) distribution analysis, we observe that there are quaternary structural changes at various substrate concentrations (Fig. A broad peak located between the monomeric and dimeric Mpro was detected at a substrate concentration of 50 lM, which suggest that Mpro is a rapidly self-associating protein [16]. This is consistent with our recent observations during routine SV experiments, which have shown that the presence of substrate can induce a quaternary structural change in Mpro [16].…”

“…At that time, the complicated data analysis problems impeded the wide spread use of this technique [9]. All the results from the AUC support the existence of substrate-induced dimerization of Mpro, which is consistent with our other studies [16]. This has changed because advances in software have made handling huge datasets only a few keyboard strokes away [3,4,13,14].…”

“…1A, the lines show the results of best-fitting to the c(s) distribution. In the presence of 200 lM substrate, Mpro exists as a single species at S = 2.3, which corresponds to the dimeric form [16]. 1B).…”

“…The detailed theoretical background and practical aspects have been thoroughly discussed [7][8][9][10][11][12]. Taking advantage of this, we have successfully applied the active enzyme centrifugation technique to the dimeric SARS-CoV Mpro in which dimerization is required for its normal functioning [15,16]. The available literature on band-forming AUC is rather scarce.…”

Differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease

“…The AUC experiments were performed on a XL-A analytical ultracentrifuge (Beckman, Fullerton, CA) using an An-50 Ti rotor [15,[17][18][19][20]. The sedimentation velocity experiments were performed using a double-sector epon charcoal-filled centerpiece at 20°C with a rotor speed of 42,000 rpm.…”