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Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains

A Corrigendum to this article was published on 13 November 2008

Abstract

Deubiquitinating enzymes (DUBs) remove ubiquitin from conjugated substrates to regulate various cellular processes. The Zn2+-dependent DUBs AMSH and AMSH-LP regulate receptor trafficking by specifically cleaving Lys 63-linked polyubiquitin chains from internalized receptors. Here we report the crystal structures of the human AMSH-LP DUB domain alone and in complex with a Lys 63-linked di-ubiquitin at 1.2 Å and 1.6 Å resolutions, respectively. The AMSH-LP DUB domain consists of a Zn2+-coordinating catalytic core and two characteristic insertions, Ins-1 and Ins-2. The distal ubiquitin interacts with Ins-1 and the core, whereas the proximal ubiquitin interacts with Ins-2 and the core. The core and Ins-1 form a catalytic groove that accommodates the Lys 63 side chain of the proximal ubiquitin and the isopeptide-linked carboxy-terminal tail of the distal ubiquitin. This is the first reported structure of a DUB in complex with an isopeptide-linked ubiquitin chain, which reveals the mechanism for Lys 63-linkage-specific deubiquitination by AMSH family members.

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Figure 1: Overall structure of the AMSH-LP DUB domain alone and in complex with K63–Ub2.
Figure 2: Proximal ubiquitin recognition.
Figure 3: Distal ubiquitin recognition.
Figure 4: Catalytic mechanism.
Figure 5: Conservation among the DUB domains of AMSH, AMSH-LP and Rpn11.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors of the AMSH-LP DUB domain and its complex with K63–Ub2 have been deposited in the Protein Data Bank with the accession codes 2ZNR and 2ZNV, respectively.

References

  1. 1

    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 

  2. 2

    Hicke, L. Protein regulation by monoubiquitin. Nature Rev. Mol. Cell Biol. 2, 195–201 (2001)

    CAS  Article  Google Scholar 

  3. 3

    Miranda, M. & Sorkin, A. Regulation of receptors and transporters by ubiquitination: new insights into surprisingly similar mechanisms. Mol. Interv. 7, 157–167 (2007)

    CAS  Article  Google Scholar 

  4. 4

    Huang, F., Kirkpatrick, D., Jiang, X., Gygi, S. & Sorkin, A. Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol. Cell 21, 737–748 (2006)

    CAS  Article  Google Scholar 

  5. 5

    Saksena, S., Sun, J., Chu, T. & Emr, S. D. ESCRTing proteins in the endocytic pathway. Trends Biochem. Sci. 32, 561–573 (2007)

    CAS  Article  Google Scholar 

  6. 6

    Amerik, A. Y. & Hochstrasser, M. Mechanism and function of deubiquitinating enzymes. Biochim. Biophys. Acta 1695, 189–207 (2004)

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

    Komada, M. Controlling receptor downregulation by ubiquitination and deubiquitination. Curr. Drug Discov. Technol. 5, 78–84 (2008)

    CAS  Article  Google Scholar 

  9. 9

    Clague, M. J. & Urbe, S. Endocytosis: the DUB version. Trends Cell Biol. 16, 551–559 (2006)

    CAS  Article  Google Scholar 

  10. 10

    Mizuno, E. et al. Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes. Mol. Biol. Cell 16, 5163–5174 (2005)

    CAS  Article  Google Scholar 

  11. 11

    McCullough, J., Clague, M. J. & Urbe, S. AMSH is an endosome-associated ubiquitin isopeptidase. J. Cell Biol. 166, 487–492 (2004)

    CAS  Article  Google Scholar 

  12. 12

    Kim, M. S., Kim, J. A., Song, H. K. & Jeon, H. STAM–AMSH interaction facilitates the deubiquitination activity in the C-terminal AMSH. Biochem. Biophys. Res. Commun. 351, 612–618 (2006)

    CAS  Article  Google Scholar 

  13. 13

    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 

  14. 14

    Reyes-Turcu, F. E. et al. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197–1208 (2006)

    CAS  Article  Google Scholar 

  15. 15

    Misaghi, S. et al. Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate. J. Biol. Chem. 280, 1512–1520 (2005)

    CAS  Article  Google Scholar 

  16. 16

    Messick, T. E. et al. Structural basis for ubiquitin recognition by the OTU1 ovarian tumor domain protein. J. Biol. Chem. 283, 11038–11049 (2008)

    CAS  Article  Google Scholar 

  17. 17

    Varadan, R., Assfalg, M., Raasi, S., Pickart, C. & Fushman, D. Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain. Mol. Cell 18, 687–698 (2005)

    CAS  Article  Google Scholar 

  18. 18

    Trempe, J. F. et al. Mechanism of Lys48-linked polyubiquitin chain recognition by the Mud1 UBA domain. EMBO J. 24, 3178–3189 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M. & Wolberger, C. Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nature Struct. Mol. Biol. 13, 915–920 (2006)

    CAS  Article  Google Scholar 

  20. 20

    Pena, V., Liu, S., Bujnicki, J. M., Luhrmann, R. & Wahl, M. C. Structure of a multipartite protein–protein interaction domain in splicing factor Prp8 and its link to Retinitis pigmentosa . Mol. Cell 25, 615–624 (2007)

    CAS  Article  Google Scholar 

  21. 21

    Zhang, L. et al. Crystal structure of the C-terminal domain of splicing factor Prp8 carrying Retinitis pigmentosa mutants. Protein Sci. 16, 1024–1031 (2007)

    CAS  Article  Google Scholar 

  22. 22

    Sanches, M., Alves, B. S., Zanchin, N. I. & Guimaraes, B. G. The crystal structure of the human Mov34 MPN domain reveals a metal-free dimer. J. Mol. Biol. 370, 846–855 (2007)

    CAS  Article  Google Scholar 

  23. 23

    Ambroggio, X. I., Rees, D. C. & Deshaies, R. J. JAMM: a metalloprotease-like zinc site in the proteasome and signalosome. PLoS Biol. 2, E2 (2004)

    Article  Google Scholar 

  24. 24

    Tran, H. J., Allen, M. D., Lowe, J. & Bycroft, M. Structure of the Jab1/MPN domain and its implications for proteasome function. Biochemistry 42, 11460–11465 (2003)

    CAS  Article  Google Scholar 

  25. 25

    Pickart, C. M. & Raasi, S. Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005)

    CAS  Article  Google Scholar 

  26. 26

    Hofmann, R. M. & Pickart, C. M. In vitro assembly and recognition of Lys-63 polyubiquitin chains. J. Biol. Chem. 276, 27936–27943 (2001)

    CAS  Article  Google Scholar 

  27. 27

    Tenno, T. et al. Structural basis for distinct roles of Lys63- and Lys48-linked polyubiquitin chains. Genes Cells 9, 865–875 (2004)

    CAS  Article  Google Scholar 

  28. 28

    Varadan, R. et al. Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling. J. Biol. Chem. 279, 7055–7063 (2004)

    CAS  Article  Google Scholar 

  29. 29

    Hurley, J. H., Lee, S. & Prag, G. Ubiquitin-binding domains. Biochem. J. 399, 361–372 (2006)

    CAS  Article  Google Scholar 

  30. 30

    Gupta, S. P. Quantitative structure–activity relationship studies on zinc-containing metalloproteinase inhibitors. Chem. Rev. 107, 3042–3087 (2007)

    CAS  Article  Google Scholar 

  31. 31

    Lipscomb, W. N. & Strater, N. Recent advances in zinc enzymology. Chem. Rev. 96, 2375–2434 (1996)

    CAS  Article  Google Scholar 

  32. 32

    Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H. & Matthews, B. W. Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues. Biochemistry 26, 8542–8553 (1987)

    CAS  Article  Google Scholar 

  33. 33

    Zhu, P. et al. A histone H2A deubiquitinase complex coordinating histone acetylation and H1 dissociation in transcriptional regulation. Mol. Cell 27, 609–621 (2007)

    CAS  Article  Google Scholar 

  34. 34

    Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007)

    ADS  CAS  Article  Google Scholar 

  35. 35

    Maytal-Kivity, V., Reis, N., Hofmann, K. & Glickman, M. H. MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function. BMC Biochem. 3, 28 (2002)

    Article  Google Scholar 

  36. 36

    Yao, T. & Cohen, R. E. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002)

    ADS  CAS  Article  Google Scholar 

  37. 37

    Verma, R. et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615 (2002)

    ADS  CAS  Article  Google Scholar 

  38. 38

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

    CAS  Article  Google Scholar 

  39. 39

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

  40. 40

    Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. D 64, 112–122 (2008)

    ADS  CAS  Article  Google Scholar 

  41. 41

    de. la Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997)

    CAS  Article  Google Scholar 

  42. 42

    Abrahams, J. P. & Leslie, A. G. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)

    CAS  Article  Google Scholar 

  43. 43

    Cowtan, K. D. & Main, P. Improvement of macromolecular electron-density maps by the simultaneous application of real and reciprocal space constraints. Acta Crystallogr. D 49, 148–157 (1993)

    CAS  Article  Google Scholar 

  44. 44

    Morris, R. J., Perrakis, A. & Lamzin, V. S. ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol. 374, 229–244 (2003)

    CAS  Article  Google Scholar 

  45. 45

    Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  PubMed  Google Scholar 

  46. 46

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

    CAS  Article  Google Scholar 

  47. 47

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

    Article  Google Scholar 

  48. 48

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

    CAS  Article  Google Scholar 

  49. 49

    Iwai, K. et al. Identification of the von Hippel–Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex. Proc. Natl Acad. Sci. USA 96, 12436–12441 (1999)

    ADS  CAS  Article  Google Scholar 

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Acknowledgements

We thank C. Toyoshima for support of this research. We thank M. Shirakawa and T. Tenno for advice about the K63–Ub2 preparation. We thank S. Kaiser for critical reading and improvement of this manuscript. We thank the beam-line staffs at NW12A of PF-AR (Tsukuba, Japan) for technical help during data collection. This work was supported by grants from MEXT to S.F. and O.N. Y.S. is supported by JSPS research fellowships for young scientists.

Author Contributions Y.S. carried out the crystallization and structure determination of the AMSH-LP DUB domain and its complex with K63–Ub2. A.Y. prepared K63–Ub2 and polyubiquitin chains with support from K.O., M.Y., O.N., K.I. and M.K. Y.S. and A.Y. carried out the in vitro DUB assays. K.I. and M.K. provided resources for K63–Ub2 preparation. A.Y., H.M. and S.F. assisted with the crystallization and structure determination. Y.S. and S.F. wrote the paper, with editing from A.Y., H.M., O.N., K.I. and M.K. S.F. and M.K. designed the research. All authors discussed the results and commented on the manuscript. S.F. supervised the work.

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Correspondence to Shuya Fukai.

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Sato, Y., Yoshikawa, A., Yamagata, A. et al. Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains. Nature 455, 358–362 (2008). https://doi.org/10.1038/nature07254

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