Protein & Cell

, Volume 1, Issue 4, pp 371–383

Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease

  • Shengnan Zhang
  • Nan Zhong
  • Fei Xue
  • Xue Kang
  • Xiaobai Ren
  • Jiaxuan Chen
  • Changwen Jin
  • Zhiyong Lou
  • Bin Xia
Research Article
  • 66 Downloads

Abstract

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.

Keywords

SARS-CoV main protease crystal structure 3D domain swapping polyprotein processing 

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References

  1. 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
  2. Anand, K., Palm, G.J., Mesters, J.R., Siddell, S.G., Ziebuhr, J., and Hilgenfeld, R. (2002). Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra α-helical domain. EMBO J 21, 3213–3224.PubMedCentralCrossRefGoogle Scholar
  3. 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
  4. Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J.R., and Hilgenfeld, R. (2003). Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs. Science 300, 1763–1767.CrossRefGoogle Scholar
  5. Bartlam, M., Yang, H., and Rao, Z. (2005). Structural insights into SARS coronavirus proteins. Curr Opin Struct Biol 15, 664–672.CrossRefGoogle Scholar
  6. Cattaruzza, S., and Perris, R. (2005). Proteoglycan control of cell movement during wound healing and cancer spreading. Matrix Biol 24, 400–417.CrossRefGoogle Scholar
  7. Chan, H.L., Tsui, S.K., and Sung, J.J. (2003). Coronavirus in severe acute respiratory syndrome (SARS). Trends Mol Med 9, 323–325.CrossRefGoogle Scholar
  8. 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
  9. Chen, H., Wei, P., Huang, C., Tan, L., Liu, Y., and Lai, L. (2006). Only one protomer is active in the dimer of SARS 3C-like proteinase. J Biol Chem 281, 13894–13898.CrossRefGoogle Scholar
  10. 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
  11. Chen, S., Jonas, F., Chen, C., and Higenfiled, R. (2010). Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode. Protein Cell 1, 59–74.CrossRefGoogle Scholar
  12. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6, 277–293.CrossRefGoogle Scholar
  13. Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132.CrossRefGoogle Scholar
  14. Fan, K., Wei, P., Feng, Q., Chen, S., Huang, C., Ma, L., Lai, B., Pei, J., Liu, Y., Chen, J., et al. (2004). Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase. J Biol Chem 279, 1637–1642.CrossRefGoogle Scholar
  15. Graziano, V., McGrath, W.J., DeGruccio, A.M., Dunn, J.J., and Mangel, W.F. (2006a). Enzymatic activity of the SARS coronavirus main proteinase dimer. FEBS Lett 580, 2577–2583.CrossRefGoogle Scholar
  16. Graziano, V., McGrath, W.J., Yang, L., and Mangel, W.F. (2006b). SARS CoV main proteinase: The monomer-dimer equilibrium dissociation constant. Biochemistry 45, 14632–14641.CrossRefGoogle Scholar
  17. Gronenborn, A.M. (2009). Protein acrobatics in pairs-dimerization via domain swapping. Curr Opin Struct Biol 19, 39–49.PubMedCentralCrossRefGoogle Scholar
  18. Hsu, W.C., Chang, H.C., Chou, C.Y., Tsai, P.J., Lin, P.I., and Chang, G.G. (2005). Critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease. J Biol Chem 280, 22741–22748.CrossRefGoogle Scholar
  19. Hu, T., Zhang, Y., Li, L., Wang, K., Chen, S., Chen, J., Ding, J., Jiang, H., and Shen, X. (2009). Two adjacent mutations on the dimer interface of SARS coronavirus 3C-like protease cause different conformational changes in crystal structure. Virology 388, 324–334.CrossRefGoogle Scholar
  20. Ivanov, D., Tsodikov, O.V., Kasanov, J., Ellenberger, T., Wagner, G., and Collins, T. (2007). Domain-swapped dimerization of the HIV-1 capsid C-terminal domain. Proc Natl Acad Sci U S A 104, 4353–4358.PubMedCentralCrossRefGoogle Scholar
  21. Johnson, B.A., and Blevins, R.A. (1994). NMR View: A computer program for the visualization and analysis of NMR data. J Biomol NMR 4, 603–614.CrossRefGoogle Scholar
  22. Koradi, R., Billeter, M., and Wuthrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. Journal of molecular graphics 14, 51–55.CrossRefGoogle Scholar
  23. Kuiken, T., Fouchier, R.A., Schutten, M., Rimmelzwaan, G.F., van Amerongen, G., van Riel, D., Laman, J.D., de Jong, T., van Doornum, G., Lim, W., et al. (2003). Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362, 263–270.CrossRefGoogle Scholar
  24. Kuo, C.-J., Chi, Y.-H., Hsu, J.T.-A., and Liang, P.-H. (2004). Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate. Biochem Biophys Res Commun 318, 862–867.CrossRefGoogle Scholar
  25. Laskowski, R., MacArthur, M., Moss, D., and Thornton, J. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26, 283–291.CrossRefGoogle Scholar
  26. Lee, T.W., Cherney, M.M., Huitema, C., Liu, J., James, K.E., Powers, J.C., Eltis, L.D., and James, M.N. (2005). Crystal structures of the main peptidase from the SARS coronavirus inhibited by a substrate-like aza-peptide epoxide. J Mol Biol 353, 1137–1151.CrossRefGoogle Scholar
  27. Leng, Q., and Bentwich, Z. (2003). A novel coronavirus and SARS. N Engl J Med 349, 709.CrossRefGoogle Scholar
  28. Libonati, M., Gotte, G., and Vottariello, F. (2008). A novel biological actions acquired by ribonuclease through oligomerization. Curr Pharm Biotechnol 9, 200–209.CrossRefGoogle Scholar
  29. Lin, P.Y., Chou, C.Y., Chang, H.C., Hsu, W.C., and Chang, G.G. (2008). Correlation between dissociation and catalysis of SARSCoV main protease. Arch Biochem Biophys 472, 34–42.CrossRefGoogle Scholar
  30. Liu, Y., and Eisenberg, D. (2002). 3D domain swapping: as domains continue to swap. Protein Sci 11, 1285–1299.PubMedCentralCrossRefGoogle Scholar
  31. Mammen, M., Choi, S.-K., and Whitesides, G.M. (1998). Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew Chem Int Ed 37, 2754–2794.CrossRefGoogle Scholar
  32. Marley, J., Lu, M., and Bracken, C. (2001). A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20, 71–75.CrossRefGoogle Scholar
  33. Matthews, B.W. (1968). Solvent content of protein crystals. J Mol Biol 33, 491–497.CrossRefGoogle Scholar
  34. McCoy, A., Grosse-Kunstleve, R., Adams, P., Winn, M., Storoni, L., and Read, R. (2007). Phaser crystallographic software. J Appl Cryst 40, 658–674.CrossRefGoogle Scholar
  35. Minor, K.H., and Peterson, C.B. (2002). Plasminogen activator inhibitor type 1 promotes the self-association of vitronectin into complexes exhibiting altered incorporation into the extracellular matrix. J Biol Chem 277, 10337–10345.CrossRefGoogle Scholar
  36. Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53, 240–255.CrossRefGoogle Scholar
  37. 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
  38. Po-Huang, L. (2006). Characterization and inhibition of SARScoronavirus main protease. Curr Top Med Chem 6, 361–176.CrossRefGoogle Scholar
  39. Ruthenburg, A.J., Li, H., Patel, D.J., and Allis, C.D. (2007). Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev Mol Cell Biol 8, 983–994.PubMedCentralCrossRefGoogle Scholar
  40. Shi, J., and Song, J. (2006). The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain. FEBS J 273, 1035–1045.CrossRefGoogle Scholar
  41. 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
  42. Shi, J., Sivaraman, J., and Song, J. (2008). Mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3C-like protease. J Virol 82, 4620–4629.PubMedCentralCrossRefGoogle Scholar
  43. 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
  44. 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
  45. Verschueren, K.H.G., Pumpor, K., Anemüller, S., Chen, S., Mesters, J.R., Hilgenfeld, R., (2008). A structural view of the inactivation of the SARS coronavirus main proteinase by benzotriazole esters. Chem Biol 15, 597–606.CrossRefGoogle Scholar
  46. Wei, P., Fan, K., Chen, H., Ma, L., Huang, C., Tan, L., Xi, D., Li, C., Liu, Y., Cao, A., et al. (2006). The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase. Biochem Biophys Res Commun 339, 865–872.CrossRefGoogle Scholar
  47. Wishart, D.S., Bigam, C.G., Yao, J., Abildgaard, F., Dyson, H.J., Oldfield, E., Markley, J.L., and Sykes, B.D. (1995). 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR 6, 135–140.CrossRefGoogle Scholar
  48. Xu, T., Ooi, A., Lee, H.C., Wilmouth, R., Liu, D.X., and Lescar, J. (2005). Structure of the SARS coronavirus main proteinase as an active C2 crystallographic dimer. Acta Crystallogr Sect F Struct Biol Cryst Commun 61, 964–966.PubMedCentralCrossRefGoogle Scholar
  49. Xue, X., Yang, H., Shen, W., Zhao, Q., Li, J., Yang, K., Chen, C., Jin, Y., Bartlam, M., and Rao, Z. (2007). Production of authentic SARSCoV M(pro) with enhanced activity: application as a novel tagcleavage endopeptidase for protein overproduction. J Mol Biol 366, 965–975.CrossRefGoogle Scholar
  50. Yamasaki, M., Li, W., Johnson, D.J., and Huntington, J.A. (2008). Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization. Nature 455, 1255–1258.CrossRefGoogle Scholar
  51. 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
  52. Yang, H., Xie, W., Xue, X., Yang, K., Ma, J., Liang, W., Zhao, Q., Zhou, Z., Pei, D., Ziebuhr, J., et al. (2005). Design of wide-spectrum inhibitors targeting coronavirus main proteases. PLoS Biol 3, e324.PubMedCentralCrossRefGoogle Scholar
  53. Yang, H., Bartlam, M., and Rao, Z. (2006). Drug design targeting the main protease, the Achilles’ heel of coronaviruses. Curr Pharm Des 12, 4573–4590.CrossRefGoogle Scholar
  54. Zhong, N., Zhang, S., Zou, P., Chen, J., Kang, X., Li, Z., Liang, C., Jin, C., and Xia, B. (2008). Without its N-finger, SARS-CoV main protease can form a novel dimer through its C-terminal domain. J Virol 82, 4227–4234.PubMedCentralCrossRefGoogle Scholar
  55. Zhong, N., Zhang, S., Xue, F., Kang, X., Zou, P., Chen, J., Liang, C., Rao, Z., Jin, C., Lou, Z., et al. (2009). C-terminal domain of SARSCoV main protease can form a 3D domain-swapped dimer. Protein Sci 18, 839–844.PubMedCentralGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Shengnan Zhang
    • 1
    • 2
  • Nan Zhong
    • 1
    • 2
  • Fei Xue
    • 3
  • Xue Kang
    • 1
    • 2
  • Xiaobai Ren
    • 1
    • 2
  • Jiaxuan Chen
    • 1
    • 4
  • Changwen Jin
    • 1
    • 2
    • 4
  • Zhiyong Lou
    • 3
  • Bin Xia
    • 1
    • 2
    • 4
  1. 1.Beijing Nuclear Magnetic Resonance CenterPeking UniversityBeijingChina
  2. 2.College of Chemistry and Molecular EngineeringPeking UniversityBeijingChina
  3. 3.Structural Biology LaboratoryTsinghua UniversityBeijingChina
  4. 4.College of Life SciencesPeking UniversityBeijingChina

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