Article | Published:

Structural basis of the specificity of USP18 toward ISG15

Nature Structural & Molecular Biology volume 24, pages 270278 (2017) | Download Citation

Abstract

Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin proteases and Ubl proteases, collectively termed DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity toward ubiquitin but specifically deconjugates the interferon-induced Ubl ISG15. To identify the molecular determinants of this specificity, we solved the crystal structures of mouse USP18 alone and in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation, thus revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15, thus providing evidence that USP18's ISG15 specificity is mediated by a small interaction interface. Our results may provide a structural basis for the development of new drugs modulating ISG15 linkage.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

References

  1. 1.

    , , & Interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin. J. Biol. Chem. 262, 11315–11323 (1987).

  2. 2.

    et al. Crystal structure of the interferon-induced ubiquitin-like protein ISG15. J. Biol. Chem. 280, 27356–27365 (2005).

  3. 3.

    & Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. EMBO J. 20, 362–371 (2001).

  4. 4.

    , , & Interferon-inducible ubiquitin E2, Ubc8, is a conjugating enzyme for protein ISGylation. Mol. Cell. Biol. 24, 9592–9600 (2004).

  5. 5.

    et al. The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-α/β-induced ubiquitin-like protein. Proc. Natl. Acad. Sci. USA 101, 7578–7582 (2004).

  6. 6.

    et al. HERC6 is the main E3 ligase for global ISG15 conjugation in mouse cells. PLoS One 7, e29870 (2012).

  7. 7.

    , , & mHERC6 is the essential ISG15 E3 ligase in the murine system. Biochem. Biophys. Res. Commun. 417, 135–140 (2012).

  8. 8.

    , , & HERC5 is an IFN-induced HECT-type E3 protein ligase that mediates type I IFN-induced ISGylation of protein targets. Proc. Natl. Acad. Sci. USA 103, 10735–10740 (2006).

  9. 9.

    , , , & Herc5, an interferon-induced HECT E3 enzyme, is required for conjugation of ISG15 in human cells. J. Biol. Chem. 281, 4334–4338 (2006).

  10. 10.

    , , & Interferon-induced ISG15 pathway: an ongoing virus-host battle. Trends Microbiol. 21, 181–186 (2013).

  11. 11.

    & The antiviral activities of ISG15. J. Mol. Biol. 425, 4995–5008 (2013).

  12. 12.

    & Emerging role of ISG15 in antiviral immunity. Cell 143, 187–190 (2010).

  13. 13.

    , , & The ISG15 conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of ISG15. Mol. Cell 38, 722–732 (2010).

  14. 14.

    et al. Proteomic identification of proteins conjugated to ISG15 in mouse and human cells. Biochem. Biophys. Res. Commun. 336, 496–506 (2005).

  15. 15.

    , , , & Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc. Natl. Acad. Sci. USA 102, 10200–10205 (2005).

  16. 16.

    et al. ISG15 modification of ubiquitin E2 Ubc13 disrupts its ability to form thioester bond with ubiquitin. Biochem. Biophys. Res. Commun. 336, 61–68 (2005).

  17. 17.

    , , , & Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme. J. Biochem. 138, 711–719 (2005).

  18. 18.

    et al. Positive regulation of interferon regulatory factor 3 activation by Herc5 via ISG15 modification. Mol. Cell. Biol. 30, 2424–2436 (2010).

  19. 19.

    et al. ISG15 enhances the innate antiviral response by inhibition of IRF-3 degradation. Cell. Mol. Biol. 52, 29–41 (2006).

  20. 20.

    , , , & Immunoregulatory properties of ISG15, an interferon-induced cytokine. Proc. Natl. Acad. Sci. USA 93, 211–215 (1996).

  21. 21.

    et al. Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency. Science 337, 1684–1688 (2012).

  22. 22.

    et al. Modification of PCNA by ISG15 plays a crucial role in termination of error-prone translesion DNA synthesis. Mol. Cell 54, 626–638 (2014).

  23. 23.

    et al. ARF and p53 coordinate tumor suppression of an oncogenic IFN-β-STAT1-ISG15 signaling axis. Cell Rep. 7, 514–526 (2014).

  24. 24.

    & IFNs, ISGylation and cancer: Cui prodest? Cytokine Growth Factor Rev. 23, 307–314 (2012).

  25. 25.

    , & Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550–563 (2009).

  26. 26.

    et al. Screen for ISG15-crossreactive deubiquitinases. PLoS One 2, e679 (2007).

  27. 27.

    et al. Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21. EMBO Rep. 12, 350–357 (2011).

  28. 28.

    et al. Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15. FEBS J. 281, 1918–1928 (2014).

  29. 29.

    , , , & UBP43 (USP18) specifically removes ISG15 from conjugated proteins. J. Biol. Chem. 277, 9976–9981 (2002).

  30. 30.

    et al. USP18 inhibits NF-κB and NFAT activation during Th17 differentiation by deubiquitinating the TAK1-TAB1 complex. J. Exp. Med. 210, 1575–1590 (2013).

  31. 31.

    et al. USP18 negatively regulates NF-κB signaling by targeting TAK1 and NEMO for deubiquitination through distinct mechanisms. Sci. Rep. 5, 12738 (2015).

  32. 32.

    et al. Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance. Proc. Natl. Acad. Sci. USA 112, 1577–1582 (2015).

  33. 33.

    & ISG15 uncut: dissecting enzymatic and non-enzymatic functions of USP18 in vivo. Cytokine 76, 569–571 (2015).

  34. 34.

    et al. Ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) in host defense against heart failure in a mouse model of virus-induced cardiomyopathy. Circulation 130, 1589–1600 (2014).

  35. 35.

    et al. USP18 lack in microglia causes destructive interferonopathy of the mouse brain. EMBO J. 34, 1612–1629 (2015).

  36. 36.

    et al. UBP43 is a novel regulator of interferon signaling independent of its ISG15 isopeptidase activity. EMBO J. 25, 2358–2367 (2006).

  37. 37.

    et al. Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome. J. Exp. Med. 213, 1163–1174 (2016).

  38. 38.

    et al. Human intracellular ISG15 prevents interferon-α/β over-amplification and auto-inflammation. Nature 517, 89–93 (2015).

  39. 39.

    et al. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054 (2002).

  40. 40.

    et al. Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation by GMP-synthetase. Mol. Cell 44, 147–159 (2011).

  41. 41.

    et al. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 24, 3747–3756 (2005).

  42. 42.

    et al. Amino-terminal dimerization, NRDP1-rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease 8 (USP8). J. Biol. Chem. 281, 38061–38070 (2006).

  43. 43.

    , & A 2.2 Å resolution structure of the USP7 catalytic domain in a new space group elaborates upon structural rearrangements resulting from ubiquitin binding. Acta Crystallogr. F Struct. Biol. Commun. 70, 283–287 (2014).

  44. 44.

    et al. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J. Am. Chem. Soc. 135, 2867–2870 (2013).

  45. 45.

    et al. Recognition of Lys48-linked di-ubiquitin and deubiquitinating activities of the SARS coronavirus papain-like protease. Mol. Cell 62, 572–585 (2016).

  46. 46.

    & Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

  47. 47.

    et al. A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187–5196 (2001).

  48. 48.

    & Regulation of mitochondrial morphology by USP30, a deubiquitinating enzyme present in the mitochondrial outer membrane. Mol. Biol. Cell 19, 1903–1911 (2008).

  49. 49.

    et al. Deubiquitinase USP37 is activated by CDK2 to antagonize APC(CDH1) and promote S phase entry. Mol. Cell 42, 511–523 (2011).

  50. 50.

    , , , & Structural basis for the ubiquitin-linkage specificity and deISGylating activity of SARS-CoV papain-like protease. PLoS Pathog. 10, e1004113 (2014).

  51. 51.

    et al. ISG15 deficiency and increased viral resistance in humans but not mice. Nat. Commun. 7, 11496 (2016).

  52. 52.

    , , , & Structural basis of recognition of interferon-α receptor by tyrosine kinase 2. Nat. Struct. Mol. Biol. 21, 443–448 (2014).

  53. 53.

    , & MultiBac: multigene baculovirus-based eukaryotic protein complex production. Curr. Protoc. Protein Sci. 52, 5.20 (2008).

  54. 54.

    , & Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 22, 1583–1587 (2004).

  55. 55.

    , , & MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem. Sci. 37, 49–57 (2012).

  56. 56.

    et al. Automated unrestricted multigene recombineering for multiprotein complex production. Nat. Methods 6, 447–450 (2009).

  57. 57.

    et al. Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins. Mol. Cell. Biol. 24, 84–95 (2004).

  58. 58.

    Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

  59. 59.

    et al. Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem. Int. Edn Engl. 49, 10149–10153 (2010).

  60. 60.

    Tricine–SDS-PAGE. Nat. Protoc. 1, 16–22 (2006).

  61. 61.

    , , , & ISG15, an interferon-stimulated ubiquitin-like protein, is not essential for STAT1 signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus. Mol. Cell. Biol. 25, 6338–6345 (2005).

  62. 62.

    XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

  63. 63.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  64. 64.

    , , & Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  65. 65.

    et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

  66. 66.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  67. 67.

    The PyMOL Molecular Graphics System, Version 1.3r1 (Schrödinger, LLC, 2010).

  68. 68.

    , , , & Jalview Version 2: a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

  69. 69.

    et al. A strategy for modulation of enzymes in the ubiquitin system. Science 339, 590–595 (2013).

  70. 70.

    , , & The DUSP-Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange. Nat. Commun. 5, 5399 (2014).

  71. 71.

    et al. Two ZnF-UBP domains in isopeptidase T (USP5). Biochemistry 51, 1188–1198 (2012).

Download references

Acknowledgements

We thank S. Ehrenfeld and A. Hausmann for assistance with cloning and expression of USP18 and D. el Atmioui for assistance with SPPS. The help of F. Garzoni in generating the insect-cell expression system is gratefully acknowledged. We thank C. Brancolini (University of Udine) and P. Boudinot (French National Institute for Agricultural Research) for providing materials. The expression-system work was financially supported by the European Community's Seventh Framework Programme (FP7/2007-2013) PCUBE. This work was further supported by grants from the Deutsche Forschungsgemeinschaft FR 1488/3-2 to G.F. and KN 590/3-2 and KN 590/1-3 to K.-P.K., and from the Dutch Technology Foundation (STW) to P.P.G. and H.O. This work was also supported by a grant from the European Research Council (ERC Grant agreement no. 281699 to H.O.). We thank the staff at beamlines X06DA and X06SA of the Swiss Light Source for excellent support. We thank the Diamond Light Source for access to beamlines I03 and I04-1 (MX9694, MX12090), which contributed to the results presented here. Finally, we thank the staff at beamlines P13 and P14 of PETRAIII for excellent support. The research leading to these results received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 283570 (for BioStruct-X).

Author information

Author notes

    • Anja Basters

    Present address: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

    • Paul P Geurink
    •  & Annika Röcker

    These authors contributed equally to this work.

    • Huib Ovaa
    • , Klaus-Peter Knobeloch
    •  & Günter Fritz

    These authors jointly supervised this work.

Affiliations

  1. Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

    • Anja Basters
    • , Annika Röcker
    • , Roya Tadayon
    • , Sandra Hess
    • , Klaus-Peter Knobeloch
    •  & Günter Fritz
  2. Department of Chemical Immunology, Leiden University Medical Center, Leiden, the Netherlands.

    • Paul P Geurink
    • , Katharina F Witting
    •  & Huib Ovaa
  3. Hermann-Staudinger Graduate School, University of Freiburg, Freiburg, Germany.

    • Roya Tadayon
  4. Structural Biology Laboratory, Elettra Sincrotrone Trieste S.C.p.A., Trieste, Italy.

    • Marta S Semrau
    •  & Paola Storici

Authors

  1. Search for Anja Basters in:

  2. Search for Paul P Geurink in:

  3. Search for Annika Röcker in:

  4. Search for Katharina F Witting in:

  5. Search for Roya Tadayon in:

  6. Search for Sandra Hess in:

  7. Search for Marta S Semrau in:

  8. Search for Paola Storici in:

  9. Search for Huib Ovaa in:

  10. Search for Klaus-Peter Knobeloch in:

  11. Search for Günter Fritz in:

Contributions

A.B., A.R., P.P.G., M.S.S. and P.S. expressed and purified proteins. A.B. and A.R. created variants and tested enzyme reactivity. A.B. crystallized the proteins and recorded X-ray diffraction data. G.F. performed data analysis. A.B. and G.F. performed model building and refinement. P.P.G. and K.F.W. created the covalent ISG15 and Ub probes and performed enzyme assays. A.B., S.H., A.R. and K.-P.K. performed cell culture experiments and immunoblot analysis. R.T. performed SPR analysis. A.B., H.O., K.-P.K. and G.F. designed experiments. A.B., K.-P.K. and G.F. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Klaus-Peter Knobeloch or Günter Fritz.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Note

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3371