Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL pro ) to cleave the virus-encoded polyproteins. We report here that the 3CL pro containing additional N-and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln The data indicate that immature 3CL pro can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL pro from the polyprotein and design of new structure-based inhibitors.

Five active metal-conjugated inhibitors (PMA, TDT, EPDTC, JMF1586 and JMF1600) bound with the 3C-like protease of severe acute respiratory syndrome (SARS)-associated coronavirus were analyzed crystallographically. The complex structures reveal two major inhibition modes: Hg 2+ -PMA is coordinated to C 44 , M 49 and Y 54 with a square planar geometry at the S3 pocket, whereas each Zn 2+ of the four zinc-inhibitors is tetrahedrally coordinated to the H 41 -C 145 catalytic dyad. For anti-SARS drug design, this Zn 2+ -centered coordination pattern would serve as a starting platform for inhibitor optimization.