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.

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

Abstract

Ubiquitin-like proteins (UBLs) such as NEDD8 are transferred to their targets by distinct, parallel, multienzyme cascades that involve the sequential action of E1, E2 and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote thioester formation between Ubc12 and NEDD8. A peptide corresponding to Ubc12's N terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1–UBA3 complex. The structure of APPBP1–UBA3– Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s. These data explain why the Ubc12-UBA3 interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Ubc12's N-terminal extension is important for function.
Figure 2: Ubc12's N terminus is involved in E1 binding.
Figure 3: Electron density maps superimposed with the Ubc12N26 peptide structure.
Figure 4: Overall architecture of the APPBP1–UBA3–Ubc12N26 complex.
Figure 5: The Ubc12N26-binding surface is conserved in UBA3s, but not in activating enzymes for other UBLs.
Figure 6: Contributions of individual residues from Ubc12's N-terminal peptide to E1 binding.
Figure 7: Minimal length requirement for the linker between the E1 docking motif and the E2 core domain in Ubc12.
Figure 8: Model for optimal positioning of Ubc12 in the E1 structure for formation of the Ubc12-NEDD8 thioester.

Accession codes

Accessions

Protein Data Bank

References

  1. Schwartz, D.C. & Hochstrasser, M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers. Trends Biochem. Sci. 28, 321–328 (2003).

    CAS  Article  PubMed  Google Scholar 

  2. Lammer, D. et al. Modification of yeast Cdc53p by the ubiquitin-related protein rub1p affects function of the SCFCdc4 complex. Genes Dev. 12, 914–926 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Liakopoulos, D., Doenges, G., Matuschewski, K. & Jentsch, S. A novel protein modification pathway related to the ubiquitin system. EMBO J. 17, 2208–2214 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Osaka, F. et al. A new NEDD8-ligating system for cullin-4A. Genes Dev. 12, 2263–2268 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Read, M.A. et al. Nedd8 modification of cul-1 activates SCF(β(TrCP))-dependent ubiquitination of IκBα. Mol. Cell. Biol. 20, 2326–2333 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Wu, K., Chen, A. & Pan, Z.Q. Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1–CUL1 complex to promote ubiquitin polymerization. J. Biol. Chem. 275, 32317–32324 (2000).

    CAS  Article  PubMed  Google Scholar 

  7. Liu, J., Furukawa, M., Matsumoto, T. & Xiong, Y. NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases. Mol. Cell 10, 1511–1518 (2002).

    CAS  Article  PubMed  Google Scholar 

  8. Zheng, J. et al. CAND1 binds to unneddylated CUL1 and regulates the formation of SCF ubiquitin E3 ligase complex. Mol. Cell 10, 1519–1526 (2002).

    CAS  Article  PubMed  Google Scholar 

  9. Osaka, F. et al. Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast. EMBO J. 19, 3475–3484 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Tateishi, K., Omata, M., Tanaka, K. & Chiba, T. The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice. J. Cell Biol. 155, 571–579 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Kurz, T. et al. Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway. Science 295, 1294–1298 (2002).

    CAS  Article  PubMed  Google Scholar 

  12. Pozo, J.C., Timpte, C., Tan, S., Callis, J. & Estelle, M. The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. Science 280, 1760–1763 (1998).

    CAS  Article  PubMed  Google Scholar 

  13. Hochstrasser, M. Evolution and function of ubiquitin-like protein-conjugation systems. Nat. Cell Biol. 2, E153–E157 (2000).

    CAS  Article  PubMed  Google Scholar 

  14. Pickart, C.M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    CAS  Article  PubMed  Google Scholar 

  15. Huang, L. et al. Structure of an E6AP–UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science 286, 1321–1326 (1999).

    CAS  Article  PubMed  Google Scholar 

  16. Johnston, S.C., Riddle, S.M., Cohen, R.E. & Hill, C.P. Structural basis for the specificity of ubiquitin C-terminal hydrolases. EMBO J. 18, 3877–3887 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Liu, Q. et al. The binding interface between an E2 (UBC9) and a ubiquitin homologue (UBL1). J. Biol. Chem. 274, 16979–16987 (1999).

    CAS  Article  PubMed  Google Scholar 

  18. Mossessova, E. & Lima, C.D. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876 (2000).

    CAS  Article  PubMed  Google Scholar 

  19. Zheng, N., Wang, P., Jeffrey, P.D. & Pavletich, N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 102, 533–539 (2000).

    CAS  Article  PubMed  Google Scholar 

  20. Brzovic, P.S., Rajagopal, P., Hoyt, D.W., King, M.C. & Klevit, R.E. Structure of a BRCA1–BARD1 heterodimeric RING–RING complex. Nat. Struct. Biol. 8, 833–837 (2001).

    CAS  Article  PubMed  Google Scholar 

  21. Hamilton, K.S. et al. Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail. Structure 9, 897–904 (2001).

    CAS  Article  PubMed  Google Scholar 

  22. Bernier-Villamor, V., Sampson, D.A., Matunis, M.J. & Lima, C.D. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108, 345–356 (2002).

    CAS  Article  PubMed  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  PubMed  Google Scholar 

  24. VanDemark, A.P. & Hill, C.P. Structural basis of ubiquitylation. Curr. Opin. Struct. Biol. 12, 822–830 (2002).

    CAS  Article  PubMed  Google Scholar 

  25. Verdecia, M.A. et al. Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase. Mol. Cell 11, 249–259 (2003).

    CAS  Article  PubMed  Google Scholar 

  26. Walden, H. et al. The structure of the APPBP1–UBA3–NEDD8–ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1. Mol. Cell 12, 1427–1437 (2003).

    CAS  Article  PubMed  Google Scholar 

  27. Furukawa, M., Zhang, Y., McCarville, J., Ohta, T. & Xiong, Y. The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1. Mol. Cell. Biol. 20, 8185–8197 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Walden, H., Podgorski, M.S. & Schulman, B.A. Insights into the ubiquitin transfer cascade from the structure of the E1 for NEDD8. Nature 422, 330–334 (2003).

    CAS  Article  PubMed  Google Scholar 

  29. Roussel, M.F., Cleveland, J.L., Shurtleff, S.A. & Sherr, C.J. Myc rescue of a mutant CSF-1 receptor impaired in mitogenic signalling. Nature 353, 361–363 (1991).

    CAS  Article  PubMed  Google Scholar 

  30. Roussel, M.F., Theodoras, A.M., Pagano, M. & Sherr, C.J. Rescue of defective mitogenic signaling by D-type cyclins. Proc. Natl. Acad. Sci. U.S.A. 92, 6837–6841 (1995).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Roussel, M.F. & Sherr, C.J. Mouse NIH 3T3 cells expressing human colony-stimulating factor 1 (CSF-1) receptors overgrow in serum-free medium containing human CSF-1 as their only growth factor. Proc. Natl. Acad. Sci. USA 86, 7924–7927 (1989).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Lake, M.W., Wuebbens, M.M., Rajagopalan, K.V. & Schindelin, H. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB–MoaD complex. Nature 414, 325–329 (2001).

    CAS  Article  PubMed  Google Scholar 

  33. Endicott, J.A., Noble, M.E. & Tucker, J.A. Cyclin-dependent kinases: inhibition and substrate recognition. Curr. Opin. Struct. Biol. 9, 738–744 (1999).

    CAS  Article  PubMed  Google Scholar 

  34. Biondi, R.M. & Nebreda, A.R. Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions. Biochem. J. 372, 1–13 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Takeda, D.Y., Wohlschlegel, J.A. & Dutta, A. A bipartite substrate recognition motif for cyclin-dependent kinases. J. Biol. Chem. 276, 1993–1997 (2001).

    CAS  Article  PubMed  Google Scholar 

  36. Yashiroda, H. & Tanaka, K. But1 and But2 proteins bind to Uba3, a catalytic subunit of E1 for neddylation, in fission yeast. Biochem. Biophys. Res. Commun. 311, 691–695 (2003).

    CAS  Article  PubMed  Google Scholar 

  37. Lyapina, S. et al. Promotion of NEDD8-CUL1 conjugate cleavage by COP9 signalosome. Science 292, 1382–1385 (2001).

    CAS  Article  PubMed  Google Scholar 

  38. Kolman, C.J., Toth, J. & Gonda, D.K. Identification of a portable determinant of cell cycle function within the carboxyl-terminal domain of the yeast CDC34 (UBC3) ubiquitin conjugating (E2) enzyme. EMBO J. 11, 3081–3090 (1992).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Silver, E.T., Gwozd, T.J., Ptak, C., Goebl, M. & Ellison, M.J. A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s. EMBO J. 11, 3091–3098 (1992).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Haldeman, M.T., Xia, G., Kasperek, E.M. & Pickart, C.M. Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element. Biochemistry 36, 10526–10537 (1997).

    CAS  Article  PubMed  Google Scholar 

  41. Morrison, A., Miller, E.J. & Prakash, L. Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae. Mol. Cell. Biol., 8, 1179–1185 (1998).

    Article  Google Scholar 

  42. Madura, K., Dohmen, R.J. & Varshavsky, A. N-recognin/Ubc2 interactions in the N-end rule pathway. J. Biol. Chem. 268, 12046–12054 (1993).

    CAS  PubMed  Google Scholar 

  43. Pan, Z.Q., Kentsis, A., Dias, D.C., Yamoah, K. & Wu, K. Nedd8 on cullin: building an expressway to protein destruction. Oncogene 23, 1985–1997 (2004).

    CAS  Article  PubMed  Google Scholar 

  44. Bencsath, K.P., Podgorski, M.S., Pagala, V.R., Slaughter, C.A. & Schulman, B.A. Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation. J. Biol. Chem. 277, 47938–47945 (2002).

    CAS  Article  PubMed  Google Scholar 

  45. Zheng, N. et al. Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002).

    CAS  Article  PubMed  Google Scholar 

  46. Bohnsack, R.N. & Haas, A.L. Conservation in the mechanism of Nedd8 activation by the human AppBp1–Uba3 heterodimer. J. Biol. Chem. 278, 26823–26830 (2003).

    CAS  Article  PubMed  Google Scholar 

  47. Hannon, G.J. et al. MaRX: an approach to genetics in mammalian cells. Science 283, 1129–1130 (1999).

    CAS  Article  PubMed  Google Scholar 

  48. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 176, 307–326 (1997).

    Article  Google Scholar 

  49. Holton, J. & Alber, T. Automated protein crystal structure determination using ELVES. Proc. Natl. Acad. Sci. USA 101, 1537–1542 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Brünger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  PubMed  Google Scholar 

  51. Whitby, F.G., Xia, G., Pickart, C.M. & Hill, C.P. Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes. J. Biol. Chem. 273, 34983–34991 (1998).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to M.S. Podgorski for initial characterization of the Ubc12ΔN mutant, to P.D. Jeffrey, N.P. Pavletich, M. Pagano, L. Hendershot, H. Walden and other members of the Schulman lab for many helpful discussions, to D.L. Minor for critical reading of the manuscript, to P.J. Murray for assistance with Figure 8, to C. Ross for crystallography support, to G. Hannon for the MaRX library and initial experiments with Ubc12 in cell proliferation assays, to S. Olsen and K. Rakestraw for expert DNA synthesis and sequencing, and to J. Tanamachi and staff at the 8.3.1 beamline at Advanced Light Source, M. Becker and staff at the X25 beamline at National Synchrotron Light Source, and staff at the SERCAT beamline at Advanced Photon Source for synchrotron support. This work was supported by American Lebanese Syrian Associated Charities, the US National Institutes of Health (P30CA21765 National Cancer Institute Cancer Center Core grant to St. Jude, R01GM69530 to B.A.S., P01CA071907 to M.F.R.), the Department of Defense (DAMD17-03-1-0420), a grant from Phillip and Elizabeth Gross, a Pew Scholar in Biomedical Sciences award to B.A.S., and a St. Jude Special Fellowship to D.H.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Brenda A Schulman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Kinetic parameters for Ubc12-NEDD8 thioester formation. (PDF 206 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Huang, D., Miller, D., Mathew, R. et al. A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8. Nat Struct Mol Biol 11, 927–935 (2004). https://doi.org/10.1038/nsmb826

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb826

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