Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease
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Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses. The main protease (Mpro) of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing. Mpro is initially produced as part of viral polyproteins and it is matured by autocleavage. Here, we report that, with the addition of an N-terminal extension peptide, Mpro can form a domain-swapped dimer. After complete removal of the extension peptide from the dimer, the mature Mpro self-assembles into a novel super-active octamer (AO-Mpro). The crystal structure of AO-Mpro adopts a novel fold with four domain-swapped dimers packing into four active units with nearly identical conformation to that of the previously reported Mpro active dimer, and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains. Compared with the previously well characterized form of Mpro, in equilibrium between inactive monomer and active dimer, the stable AO-Mpro exhibits much higher proteolytic activity at low concentration. As all eight active sites are bound with inhibitors, the polyvalent nature of the interaction between AO-Mpro and its polyprotein substrates with multiple cleavage sites, would make AO-Mpro functionally much more superior than the Mpro active dimer for polyprotein processing. Thus, during the initial period of SARS-CoV infection, this novel active form AOMpro should play a major role in cleaving polyproteins as the protein level is extremely low. The discovery of AOMpro provides new insights about the functional mechanism of Mpro and its maturation process.
KeywordsSARS-CoV main protease crystal structure 3D domain swapping polyprotein processing
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- Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.-W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002). PHENIX: building new software for automated crystallographic structure determination Acta Crystallogr D 58, 1948–1954.CrossRefGoogle Scholar
- Anand, K., Yang, H., Bartlam, M., Rao, Z. & Hilgenfeld, R. (2005). Coronavirus main proteinase: target for antiviral drug therapy. In: Coronaviruses with special emphasis on first insights concerning SARS, A. Schmidt, M.H. Wolff, and O.F. Weber, ed. (Switzerland, Basel; Birkhauser Verlag). pp. 173–199.CrossRefGoogle Scholar
- Chen, S., Chen, L., Tan, J., Chen, J., Du, L., Sun, T., Shen, J., Chen, K., Jiang, H., and Shen, X. (2005). Severe acute respiratory syndrome coronavirus 3C-like proteinase N terminus is indispensable for proteolytic activity but not for enzyme dimerization. Biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations. J Biol Chem 280, 164–173.CrossRefGoogle Scholar
- Chen, S., Hu, T., Zhang, J., Chen, J., Chen, K., Ding, J., Jiang, H., and Shen, X. (2008). Mutation of Gly11 on the dimer interface results in the complete crystallographic dimer dissociation of SARS-CoV 3CLpro: Crystal structure with molecular dynamics simulations. J Biol Chem 283, 554–564.CrossRefGoogle Scholar
- Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. In Macromolecular Crystallography, part A, C.W. Carter Jr., and R.M. Sweet, eds. (Academic Press), pp. 307–326.Google Scholar
- Shi, J., Wei, Z., and Song, J. (2004). Dissection study on the severe acute respiratory syndrome 3C-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors. J Biol Chem 279, 24765–24773.CrossRefGoogle Scholar
- Snijder, E.J., Bredenbeek, P.J., Dobbe, J.C., Thiel, V., Ziebuhr, J., Poon, L.L., Guan, Y., Rozanov, M., Spaan, W.J., and Gorbalenya, A.E. (2003). Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331, 991–1004.CrossRefGoogle Scholar
- Tan, J., Verschueren, K.H., Anand, K., Shen, J., Yang, M., Xu, Y., Rao, Z., Bigalke, J., Heisen, B., Mesters, J.R., et al. (2005). pH-dependent conformational flexibility of the SARS-CoV main proteinase (M(pro)) dimer: molecular dynamics simulations and multiple X-ray structure analyses. J Mol Biol 354, 25–40.CrossRefGoogle Scholar
- Yang, H., Yang, M., Ding, Y., Liu, Y., Lou, Z., Zhou, Z., Sun, L., Mo, L., Ye, S., Pang, H., et al. (2003). The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor. Proc Natl Acad Sci U S A 100, 13190–13195.PubMedCentralCrossRefGoogle Scholar