Virologica Sinica

, Volume 31, Issue 4, pp 288–299 | Cite as

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin

  • Jian Lei
  • Rolf HilgenfeldEmail author
Research Article


The papain-like protease (PLpro) of Middle-East respiratory syndrome coronavirus (MERS-CoV) has proteolytic, deubiquitinating, and deISGylating activities. The latter two are involved in the suppression of the antiviral innate immune response of the host cell. To contribute to an understanding of this process, we present here the X-ray crystal structure of a complex between MERS-CoV PLpro and human ubiquitin (Ub) that is devoid of any covalent linkage between the two proteins. Five regions of the PLpro bind to two areas of the Ub. The C-terminal five residues of Ub, RLRGG, are similar to the P5–P1 residues of the polyprotein substrates of the PLpro and are responsible for the major part of the interaction between the two macromolecules. Through sitedirected mutagenesis, we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PLpro. The enzyme appears not to be optimized for catalytic efficiency; thus, replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities. Ubiquitin binding by MERS-CoV PLpro involves remarkable differences compared to the corresponding complex with SARS-CoV PLpro. The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PLpro.


coronavirus Middle-East respiratory syndrome (MERS) papain-like protease ubiquitin deubiquitinase 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

12250_2016_3742_MOESM1_ESM.pdf (193 kb)
Supplementary material, approximately 194 KB.


  1. Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, Alabdullatif ZN, Assad M, Almulhim A, Makhdoom H, Madani H, Alhakeem R, Al-Tawfiq JA, Cotton M, Watson SJ, Kellam P, Zumla AI, Memish ZA, KSA MERSCoV Investigation Team. 2013. Hospital outbreak of Middle East respiratory syndrome coronavirus. N Engl J Med, 369: 407–416.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Baez-Santos YM, Barraza SJ, Wilson MW, Agius MP, Mielech AM, Davis NM, Baker SC, Larsen SD, Mesecar AD. 2014a. Xray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases. J Med Chem, 57: 2393–2412.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Baez-Santos YM, Mielech AM, Deng X, Baker S, Mesecar AD. 2014b. Catalytic function and substrate specificity of the PLpro domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). J Virol, 88: 12511–12527.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bailey-Elkin BA, Knaap RC, Johnson GG, Dalebout TJ, Ninaber DK, van Kasteren PB, Bredenbeek PJ, Snijder EJ, Kikkert M, Mark BL. 2014. Crystal structure of the Middle East respiratory syndrome coronavirus (MERS-CoV) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression. J Biol Chem, 289: 34667–34682.CrossRefPubMedPubMedCentralGoogle Scholar
  5. Barretto N, Jukneliene D, Ratia K, Chen Z, Mesecar AD, Baker SC. 2005. The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J Virol, 79: 15189–15198.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bekes M, Rut W, Kasperkiewicz P, Mulder MP, Ovaa H, Drag M, Lima CD, Huang TT. 2015. SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme. Biochem J, 468: 215–226.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Butler D. 2015. South Korean MERS outbreak spotlights lack of research. Nature, 522: 139–140.CrossRefPubMedGoogle Scholar
  8. Chang CK, Hou MH, Chang CF, Hsiao CD, Huang TH. 2014. The SARS coronavirus nucleocapsid protein–forms and functions. Antiviral Res, 103: 39–50.CrossRefPubMedGoogle Scholar
  9. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. 2010. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr, 66: 12–21.CrossRefPubMedGoogle Scholar
  10. Chen Y, Cai H, Pan J, Xiang N, Tien P, Ahola T, Guo D. 2009. Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc Natl Acad Sci USA, 106: 3484–3489.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chou CY, Lai HY, Chen HY, Cheng SC, Cheng KW, Chou YW. 2014. Structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease. Acta Crystallogr D Biol Crystallogr, 70: 572–581.CrossRefPubMedGoogle Scholar
  12. Dikic I, Wakatsuki S, Walters KJ. 2009. Ubiquitin-binding domains–from structures to functions. Nat Rev Mol Cell Biol, 10: 659–671.CrossRefPubMedGoogle Scholar
  13. Drosten C, Günther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Müller S, Rickerts V, Stürmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med, 348: 1967–1976.CrossRefPubMedGoogle Scholar
  14. Eckerle I, Müller MA, Kallies S, Gotthardt DN, Drosten C. 2013. In-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during Middle East Respiratory Syndrome (MERS) coronavirus infection. Virol J, 10: 359.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Emsley P, Lohkamp B, Scott WG, Cowtan K. 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr, 66: 486–501.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fetics SK, Guterres H, Kearney BM, Buhrman G, Ma B, Nussinov R, Mattos C. 2015. Allosteric effects of the oncogenic RasQ61L mutant on Raf-RBD. Structure, 23: 505–516.CrossRefPubMedGoogle Scholar
  17. Frieman M, Ratia K, Johnston RE, Mesecar AD, Baric RS. 2009. Severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of IRF3 and NF-?B signaling. J Virol, 83: 6689–6705.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hale BG, Randall RE, Ortin J, Jackson D. 2008. The multifunctional NS1 protein of influenza A viruses. J Gen Virol, 89: 2359–2376.CrossRefPubMedGoogle Scholar
  19. Hamre D, Procknow JJ. 1966. A new virus isolated from the human respiratory tract. Proc Soc Exp Biol Med, 121: 190–193.CrossRefPubMedGoogle Scholar
  20. Harcourt BH, Jukneliene D, Kanjanahaluethai A, Bechill J, Severson KM, Smith CM, Rota PA, Baker SC. 2004. Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity. J Virol, 78: 13600–13612.CrossRefPubMedPubMedCentralGoogle Scholar
  21. Headd JJ, Echols N, Afonine PV, Grosse-Kunstleve RW, Chen VB, Moriarty NW, Richardson DC, Richardson JS, Adams PD. 2012. Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution. Acta Crystallogr D Biol Crystallogr, 68: 381–390.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hilgenfeld R. 2014. From SARS to MERS: Crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS J, 281: 4085–4096.CrossRefPubMedGoogle Scholar
  23. Hilgenfeld R, Peiris M. 2013. From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses. Antiviral Res, 100: 286–295.CrossRefPubMedGoogle Scholar
  24. Hu M, Li P, Song L, Jeffrey PD, Chenova TA, Wilkinson KD, Cohen RE, Shi Y. 2005. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J, 24: 3747–3756.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kabsch W. 2010. XDS. Acta Crystallogr D Biol Crystallogr, 66: 125–132.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Krissinel E, Henrick K. 2007. Inference of macromolecular assemblies from crystalline state. J Mol Biol, 372: 774–797.CrossRefPubMedGoogle Scholar
  27. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, De Risi J, Yang JY, Cox N, Hughes JM, Le-Duc JW, Bellini WJ, Anderson LJ, SARS Working Group. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med, 348: 1953–1966.CrossRefPubMedGoogle Scholar
  28. Kuiken T, Fouchier RA, Schutten M, Rimmelzwaan GF, van Amerongen G, van Riel D, Laman JD, de Jong T, van Doornum G, Lim W, Ling AE, Chan PK, Tam JS, Zambon MC, Gopal R, Drosten C, van der Werf S, Escriou N, Manuguerra JC, Stöhr K, Peiris JS, Osterhaus AD. 2003. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet, 362: 263–270.CrossRefPubMedGoogle Scholar
  29. Lee H, Lei H, Santarsiero BD, Gatuz JL, Cao S, Rice AJ, Patel K, Szypulinski MZ, Ojeda I, Ghosh AK, Johnson ME. 2015. Inhibitor recognition specificity of MERS-CoV papain-like protease may differ from that of SARS-CoV. ACS Chem Biol, 10: 1456–1465.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lei J, Mesters JR, Drosten C, Anemüller S, Ma Q, Hilgenfeld R. 2014. Crystal structure of the papain-like protease of MERScoronavirus reveals unusual, potentially druggable active-site features. Antiviral Res, 109: 72–82.CrossRefPubMedGoogle Scholar
  31. Liu YC, Penninger J, Karin M. 2005. Immunity by ubiquitylation: a reversible process of modification. Nat Rev Immunol, 5: 941–952.CrossRefPubMedGoogle Scholar
  32. Maringer K, Fernandez-Sesma A. 2014. Message in a bottle: lessons learned from antagonism of STING signalling during RNA virus infection. Cytokine Growth Factor Rev, 25: 669–679.CrossRefPubMedPubMedCentralGoogle Scholar
  33. McIntosh K, Becker WB, Chanock RM. 1967. Growth in suckling- mouse brain of “IBV-like” viruses from patients with upper respiratory tract disease. Proc Natl Acad Sci USA, 58: 2268–2273.CrossRefPubMedPubMedCentralGoogle Scholar
  34. Mielech AM, Kilianski A, Baez-Santos YM, Mesecar AD, Baker SC. 2014. MERS-CoV papain-like protease has delSGylating and deubiquitinating activities. Virology, 450-451: 64–70.CrossRefPubMedGoogle Scholar
  35. Mielech AM, Deng X, Chen Y, Kindler E, Wheeler DL, Mesecar AD, Thiel V, Perlman S, Baker SC. 2015. Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis. J Virol, 89: 4907–4917.CrossRefPubMedPubMedCentralGoogle Scholar
  36. Mueller U, Darowski N, Fuchs MR, Förster R, Hellmig M, Paithankar KS, Pühringer S, Steffien M, Zocher G, Weiss MS. 2012. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. J Synchrotron Radiat, 19: 442–449.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY, Lim W, Nicholls J, Yee WK, Yan WW, Cheung MT, Cheng VC, Chan KH, Tsang DN, Yung RW, Ng TK, Yuen KY, SARS Study Group. 2003. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet, 361: 1319–1325.CrossRefPubMedGoogle Scholar
  38. Pfoh R, Lacdao IK, Georges AA, Capar A, Zheng H, Frappier L, Saridakis V. 2015. Crystal structure of USP7 ubiquitin-like domains with an ICP0 peptide reveals a novel mechanism used by viral and cellular proteins to target USP7. PLoS Pathog, 11: e1004950.CrossRefGoogle Scholar
  39. Ratia K, Kilianski A, Baez-Santos YM, Baker SC, Mesecar AD. 2014. Structural basis for the ubiquitin-linkage specificity and deISGylating activity of SARS-CoV papain-like protease. PLoS Pathog, 10: e1004113.CrossRefGoogle Scholar
  40. Vagin A, Teplyakov A. 2010. Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr, 66: 22–25.CrossRefPubMedGoogle Scholar
  41. van der Hoek L, Pyrc K, Jebbink MF, Vermeulen-Oost W, Berkhout RJ, Wolthers KC, Wertheim-van Dillen PM, Kaandorp J, Spaargaren J, Berkhout B. 2004. Identification of a new human coronavirus. Nat Med, 10: 368–373.CrossRefPubMedGoogle Scholar
  42. Vijay-Kumar S, Bugg CE, Cook WJ. 1987. Structure of ubiquitin refined at 1.8 Å resolution. J Mol Biol, 194: 531–534.CrossRefPubMedGoogle Scholar
  43. Wang H, Xue S, Yang H, Chen C. 2016. Recent progress in the discovery of inhibitors targeting coronavirus proteases. Virol Sin, 31: 24–30.CrossRefPubMedGoogle Scholar
  44. Weiss MS, Hilgenfeld R. 1997. On the use of the merging R factor as a quality indicator for X-ray data. J Appl Cryst, 30: 203–205.CrossRefGoogle Scholar
  45. Wojdyla JA, Manolaridis I, van Kasteren PB, Kikkert M, Snijder EJ, Gorbalenya AE, Tucker PA. 2010. Papain-like protease 1 from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates. J Virol, 84: 10063–10073.CrossRefPubMedPubMedCentralGoogle Scholar
  46. Woo PC, Lau SK, Chu CM, Chan KH, Tsoi HW, Huang Y, Wong BH, Poon RW, Cai JJ, Luk WK, Poon LL, Wong SS, Guan Y, Peiris JS, Yuen KY. 2005. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J Virol, 79: 884–895.CrossRefPubMedPubMedCentralGoogle Scholar
  47. Yang X, Chen X, Bian G, Tu J, Xing Y, Wang Y, Chen Z. 2013. Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease. J Gen Virol, 95: 614–626.CrossRefPubMedGoogle Scholar
  48. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med, 367: 1814–1820.CrossRefPubMedGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2016

Authors and Affiliations

  1. 1.Institute of Biochemistry, Center for Structural and Cell Biology in MedicineUniversity of LübeckLübeckGermany
  2. 2.German Center for Infection Research (DZIF)University of LübeckLübeckGermany

Personalised recommendations