Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structures of CYLD USP with Met1- or Lys63-linked diubiquitin reveal mechanisms for dual specificity

Abstract

The tumor suppressor CYLD belongs to a ubiquitin (Ub)-specific protease (USP) family and specifically cleaves Met1- and Lys63-linked polyubiquitin chains to suppress inflammatory signaling pathways. Here, we report crystal structures representing the catalytic states of zebrafish CYLD for Met1- and Lys63-linked Ub chains and two distinct precatalytic states for Met1-linked chains. In both catalytic states, the distal Ub is bound to CYLD in a similar manner, and the scissile bond is located close to the catalytic residue, whereas the proximal Ub is bound in a manner specific to Met1- or Lys63-linked chains. Further structure-based mutagenesis experiments support the mechanism by which CYLD specifically cleaves both Met1- and Lys63-linked chains and provide insight into tumor-associated mutations of CYLD. This study provides new structural insight into the mechanisms by which USP family deubiquitinating enzymes recognize and cleave Ub chains with specific linkage types.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Crystal structures of the CYLDΔBbox USP domain in Met1 and Lys63 catalytic states.
Figure 2: Recognition of Ubdist and Ubprox by zCYLDΔBbox in the Met1 and Lys63 catalytic states.
Figure 3: Conformational rearrangement of the CYLD USP domain upon binding to Ub chains.
Figure 4: Suppression of JNK and NF-κB pathways by hCYLD.
Figure 5: Structural comparison between CYLD and USP7.
Figure 6: Cleavage mechanism for Met1 chains.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Blake, P.W. & Toro, J.R. Update of cylindromatosis gene (CYLD) mutations in Brooke-Spiegler syndrome: novel insights into the role of deubiquitination in cell signaling. Hum. Mutat. 30, 1025–1036 (2009).

    CAS  Article  Google Scholar 

  2. van den Ouweland, A.M. et al. Identification of a large rearrangement in CYLD as a cause of familial cylindromatosis. Fam. Cancer 10, 127–132 (2011).

    Article  Google Scholar 

  3. Bignell, G.R. et al. Identification of the familial cylindromatosis tumour-suppressor gene. Nat. Genet. 25, 160–165 (2000).

    CAS  Article  Google Scholar 

  4. Trompouki, E. et al. CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activation by TNFR family members. Nature 424, 793–796 (2003).

    CAS  Article  Google Scholar 

  5. Brummelkamp, T.R., Nijman, S.M., Dirac, A.M. & Bernards, R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature 424, 797–801 (2003).

    CAS  Article  Google Scholar 

  6. Kovalenko, A. et al. The tumour suppressor CYLD negatively regulates NF-κB signalling by deubiquitination. Nature 424, 801–805 (2003).

    CAS  Article  Google Scholar 

  7. Nijman, S.M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005).

    CAS  Article  Google Scholar 

  8. Komander, D. et al. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. Mol. Cell 29, 451–464 (2008).

    CAS  Article  Google Scholar 

  9. Komander, D. et al. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep. 10, 466–473 (2009).

    CAS  Article  Google Scholar 

  10. Virdee, S., Ye, Y., Nguyen, D.P., Komander, D. & Chin, J.W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat. Chem. Biol. 6, 750–757 (2010).

    CAS  Article  Google Scholar 

  11. Bremm, A., Freund, S.M. & Komander, D. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nat. Struct. Mol. Biol. 17, 939–947 (2010).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  13. Trang, V.H. et al. Nonenzymatic polymerization of ubiquitin: single-step synthesis and isolation of discrete ubiquitin oligomers. Angew. Chem. Int. Edn Engl. 51, 13085–13088 (2012).

    CAS  Article  Google Scholar 

  14. Hayden, M.S. & Ghosh, S. NF-κB, the first quarter-century: remarkable progress and outstanding questions. Genes Dev. 26, 203–234 (2012).

    CAS  Article  Google Scholar 

  15. Iwai, K. Diverse ubiquitin signaling in NF-κB activation. Trends Cell Biol. 22, 355–364 (2012).

    CAS  Article  Google Scholar 

  16. Glickman, M.H. & Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).

    CAS  Article  Google Scholar 

  17. Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).

    CAS  Article  Google Scholar 

  18. Adhikari, A., Xu, M. & Chen, Z.J. Ubiquitin-mediated activation of TAK1 and IKK. Oncogene 26, 3214–3226 (2007).

    CAS  Article  Google Scholar 

  19. Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nat. Cell Biol. 11, 123–132 (2009).

    CAS  Article  Google Scholar 

  20. Rahighi, S. et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation. Cell 136, 1098–1109 (2009).

    CAS  Article  Google Scholar 

  21. Sato, Y. et al. Specific recognition of linear ubiquitin chains by the Npl4 zinc finger (NZF) domain of the HOIL-1L subunit of the linear ubiquitin chain assembly complex. Proc. Natl. Acad. Sci. USA 108, 20520–20525 (2011).

    CAS  Article  Google Scholar 

  22. Tokunaga, F. et al. Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation. EMBO J. 31, 3856–3870 (2012).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  24. Almeida, S., Maillard, C., Itin, P., Hohl, D. & Huber, M. Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity. J. Invest. Dermatol. 128, 587–593 (2008).

    CAS  Article  Google Scholar 

  25. Takiuchi, T. et al. Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN. Genes Cells 19, 254–272 (2014).

    CAS  Article  Google Scholar 

  26. Renatus, M. et al. Structural basis of ubiquitin recognition by the deubiquitinating protease USP2. Structure 14, 1293–1302 (2006).

    CAS  Article  Google Scholar 

  27. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004).

    CAS  Article  Google Scholar 

  28. Sato, Y. et al. Structural basis for specific cleavage of Lys63-linked polyubiquitin chains. Nature 455, 358–362 (2008).

    CAS  Article  Google Scholar 

  29. Juang, Y.C. et al. OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function. Mol. Cell 45, 384–397 (2012).

    CAS  Article  Google Scholar 

  30. Wiener, R., Zhang, X., Wang, T. & Wolberger, C. The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 483, 618–622 (2012).

    CAS  Article  Google Scholar 

  31. Licchesi, J.D. et al. An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains. Nat. Struct. Mol. Biol. 19, 62–71 (2012).

    CAS  Article  Google Scholar 

  32. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  Article  Google Scholar 

  33. Collaborative Computational Project, Number 4. The CCP4 Suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  34. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    CAS  Article  Google Scholar 

  35. Vijay-Kumar, S., Bugg, C.E. & Cook, W.J. Structure of ubiquitin refined at 1.8 Å resolution. J. Mol. Biol. 194, 531–544 (1987).

    CAS  Article  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  37. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank S. Kaiser for critically reading and improving this manuscript. We also thank the beamline staff at NW12A, BL-1A, BL-5A and BL17A of the Photon Factory (Tsukuba, Japan) and BL32XU and BL41XU of SPring-8 (Hyogo, Japan) for technical help during data collection. This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas 22121003 (S.F.), 22117002 (J.I.), 22117003 (M.T.), 22117006 (F.T.), 25117711 (Y. Sato) and 25112505 (Y. Sato), Grant-in-Aid for Young Scientists (A) 24687012 (Y. Sato), Grant-in-Aid for Scientific Research (A) 24247014 (S.F.) and CREST, JST (S.F.).

Author information

Authors and Affiliations

Authors

Contributions

Y. Sato designed and performed all experiments except the cell-based assays with hCYLD and wrote the paper. E.G. prepared the expression vectors of hCYLD and performed the DUB assay of hCYLD and the luciferase assay with LUBAC. Y. Shibata performed the luciferase assay with TRAF6. Y.K. performed the experiment for detection of JNK1 phosphorylation. A.Y., S.G.-I. and K.K. discussed the results. J.I., M.T. and F.T. designed and supervised the cell-based assays. S.F. supervised the work, designed the experiments and wrote the paper.

Corresponding author

Correspondence to Shuya Fukai.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of zCYLDΔBbox.

(a) Linkage specificity of zCYLDΔBbox. Ub2 species (10 μM) were incubated in the presence of 10 μM zCYLDΔBbox at 37 °C for 5 and 90 min and analyzed by tricine SDS-PAGE with Coomassie brilliant blue staining.

(b) Superposition of zCYLDΔBbox in the Met1 catalytic state and hCYLD (containing B-box) in the apo state (PDB:2VHF). The drawing scheme is the same as that in Fig. 1. The USP domain and the B-box domain of hCYLD are colored orange and yellow, respectively. In the Met1 catalytic state, both Ubdist and Ubprox are more than 12 Å from the likely position of the B-box domain, as estimated from the apo structure of the hCYLD USP domain. This is consistent with the B-box domain being dispensable for Met1- and Lys63-linkage-specific DUB activities of CYLD.

Supplementary Figure 2 Kinetic analyses of zCYLDΔBbox, Met1- and Lys63-Ub2 mutants.

Plots of initial velocity as a function of Met1- or Lys63-Ub2 concentration. Error bars represent standard deviations in three independent experiments. The data were fitted to the Michaelis-Menten kinetic model.

(a) Cleavage of Met1-Ub2 by mutant zCYLDΔBbox

(b) Cleavage of mutant Met1-Ub2 by zCYLDΔBbox

(c) Cleavage of Lys63-Ub2 by mutant zCYLDΔBbox

(d) Cleavage of mutant Lys63-Ub2 by zCYLDΔBbox

(e) DUB activity (kcat/KM) of mutant zCYLDΔBbox and Met1- and Lys63-Ub2. The experiments were carried out three times for each mutant except the Q711K mutant of zCYLDΔBbox (twice). Data are presented as mean ± SD. N.D. indicates non-detectable level.

Supplementary Figure 3 Structural details of zCYLDΔBbox in catalytic and precatalytic states.

(a) Comparison between the Met1 and Lys63 catalytic states. The structures in both states are superposed using zCYLDΔBbox as the reference. The drawing scheme is the same as that in Fig. 1, except that Ubprox in the Lys63 catalytic state is colored yellow.

(b) Comparison between the Met1 precatalytic states I and II. The structures of zCYLDΔBbox in these two states are superposed. Ubdist and zCYLDΔBbox are colored cyan and gray, respectively. Ubprox moieties in the Met1 precatalytic states I and II are colored green and magenta, respectively.

(c) Close-up views around Glu64 of Ubprox in the Met1 and Lys63 catalytic states. The drawing scheme is the same as that in Fig. 1.

(d) Cleavage of 10 μM Lys11- and Lys48-Ub2 by 40 μM wild-type or mutant zCYLDΔBbox at 37 °C for 90 min. Samples were analyzed by SDS-PAGE using SuperSepTM Ace, 5-20% (Wako) with Coomassie brilliant blue staining.

(e) Close-up views around catalytic triad of CYLD in the Met1 precatalytic states I and II. The drawing scheme is the same as that in Fig. 1.

Supplementary Figure 4 Sequence alignment of CYLD USP domains from representative organisms.

100% and more than 75% identical residues are highlighted with red backgrounds and red characters, respectively. Residues that interact with the Ubdist core, Ubdist tail and Ubprox are indicated by cyan squares, cyan triangles and pink squares, respectively. Filled squares and triangles indicate that the side chains are involved in the interaction, whereas open symbols indicate that the main chains but not side chains are involved in the interaction. Residues that form the catalytic triad are indicated by yellow stars. The residue number and secondary structure of zCYLDΔBbox are shown above the alignment.

Supplementary Figure 5 DUB activity of full-length hCYLD.

The full-length hCYLD was overexpressed in HEK293T cells.

(a) Linkage specificity of full-length hCYLD. All possible linkage types of Ub2 were cleaved by the full-length hCYLD.

(b) Cleavage of Met1 and Lys63 chains by full-length wild-type or mutant hCYLD

Supplementary Figure 6 Sequence and structure alignments of USP domains of CYLD, USP2, USP7 and USP21.

Structure alignment is based on superposition of the USP2, USP7 or USP21 complex on the zCYLD complex in the Met1 catalytic state using the PDBeFold server with the zCYLD USP domain as the reference (Cα r.m.s.d. values are 2.64, 2.70 and 2.72 Å over 219 residues in total, respectively).

(a) Structure-based sequence alignment of USP domains of zCYLD, human USP2 (PDB:2HD5), human USP7 (PDB:1NBF) and human USP21 (PDB:2Y5B). The drawing scheme is the same as that in Supplementary Fig. 4. Blue bars above the alignment correspond to the CYLD-specific truncations and insertion. The secondary structures of zCYLD and hUSP7 are shown above the alignment.

(b) Structure alignment of Ub molecules bound to USP2, USP7 and USP21 (colored yellow, green and blue, respectively). The USP domain of USP7 (colored red) is also shown as a representative USP domain.

(c) Structure alignment of Ub(dist) molecules bound to zCYLDΔBbox and USP7 (colored cyan and green, respectively). Ubprox and zCYLDΔBbox (colored pink and grey, respectively) are also shown.

(d) Structure alignment of zCYLDΔBbox in the Met1 catalytic state and USP7. The coloring scheme is the same as that in Supplementary Fig. 6b,c. Truncations (Fingers subdomain, the β-sheet intervening between β4 and β5 and the loop connecting β6 and β7) and an insertion (β9-β10 sheet) are indicated by blue circles.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 7143 kb)

Supplementary Data Sets 1–4

Uncropped western blots, intact Ub chains in crystals and raw data used for kinetic analysis (PDF 2810 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sato, Y., Goto, E., Shibata, Y. et al. Structures of CYLD USP with Met1- or Lys63-linked diubiquitin reveal mechanisms for dual specificity. Nat Struct Mol Biol 22, 222–229 (2015). https://doi.org/10.1038/nsmb.2970

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2970

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing