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Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex

Abstract

SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination1,2. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases1,2. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein3,4,5,6. Here we describe the 3.0-Å crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO–E2-thioester in an optimal orientation to enhance conjugation.

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Figure 1: Structure of SUMO–RanGAP1–Ubc9–Nup358/RanBP2 complex.
Figure 2: E2 active site in complex with RanGAP1–SUMO-1.
Figure 3: Nup358/RanBP2 sequence alignment and E2–E3–SUMO-1 structure.
Figure 4: Nup358/RanBP2 activities.

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References

  1. Johnson, E. S. Protein modification by SUMO. Annu. Rev. Biochem. 73, 355–382 (2004)

    Article  CAS  Google Scholar 

  2. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998)

    Article  CAS  Google Scholar 

  3. Matunis, M. J., Coutavas, E. & Blobel, G. A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135, 1457–1470 (1996)

    Article  CAS  Google Scholar 

  4. Mahajan, R., Delphin, C., Guan, T., Gerace, L. & Melchior, F. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97–107 (1997)

    Article  CAS  Google Scholar 

  5. Saitoh, H., Pu, R., Cavenagh, M. & Dasso, M. RanBP2 associates with Ubc9p and a modified form of RanGAP1. Proc. Natl Acad. Sci. USA 94, 3736–3741 (1997)

    Article  CAS  ADS  Google Scholar 

  6. Zhang, H., Saitoh, H. & Matunis, M. J. Enzymes of the SUMO modification pathway localize to filaments of the nuclear pore complex. Mol. Cell. Biol. 22, 6498–6508 (2002)

    Article  CAS  Google Scholar 

  7. Deshaies, R. J. SCF and Cullin/Ring H2-based ubiquitin ligases. Annu. Rev. Cell Dev. Biol. 15, 435–467 (1999)

    Article  CAS  Google Scholar 

  8. Huibregtse, J. M., Scheffner, M., Beaudenon, S. & Howley, P. M. A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl Acad. Sci. USA 92, 2563–2567 (1995)

    Article  CAS  ADS  Google Scholar 

  9. 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)

    Article  CAS  Google Scholar 

  10. Johnson, E. S. & Gupta, A. A. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell 106, 735–744 (2001)

    Article  CAS  Google Scholar 

  11. Kahyo, T., Nishida, T. & Yasuda, H. Involvement of PIAS1 in the sumoylation of tumor suppressor p53. Mol. Cell 8, 713–718 (2001)

    Article  CAS  Google Scholar 

  12. Pichler, A., Gast, A., Seeler, J. S., Dejean, A. & Melchior, F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108, 109–120 (2002)

    Article  CAS  Google Scholar 

  13. Kagey, M. H., Melhuish, T. A. & Wotton, D. The polycomb protein Pc2 is a SUMO E3. Cell 113, 127–137 (2003)

    Article  CAS  Google Scholar 

  14. Yokoyama, N. et al. A giant nucleopore protein that binds Ran/TC4. Nature 376, 184–188 (1995)

    Article  CAS  ADS  Google Scholar 

  15. Wu, J., Matunis, M. J., Kraemer, D., Blobel, G. & Coutavas, E. Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region. J. Biol. Chem. 270, 14209–14213 (1995)

    Article  CAS  Google Scholar 

  16. Joseph, J., Liu, S. T., Jablonski, S. A., Yen, T. J. & Dasso, M. The RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions in vivo . Curr. Biol. 14, 611–617 (2004)

    Article  CAS  Google Scholar 

  17. Saitoh, H., Pizzi, M. D. & Wang, J. Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358. J. Biol. Chem. 277, 4755–4763 (2002)

    Article  CAS  Google Scholar 

  18. Pichler, A., Knipscheer, P., Saitoh, H., Sixma, T. K. & Melchior, F. The RanBP2 SUMO E3 ligase is neither HECT nor RING type. Nature Struct. Mol. Biol. 11, 984–991 (2004)

    Article  CAS  Google Scholar 

  19. Tatham, M. H. et al. Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection. Nature Struct. Mol. Biol. 12, 67–74 (2004)

    Article  Google Scholar 

  20. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)

    Article  CAS  Google Scholar 

  21. Lois, L. M. & Lima, C. D. Structures of the Small Ubiquitin-like MOdifier E1 activating enzyme provide insights into SUMO activation and the mechanism for E2 recruitment to E1. EMBO J. 24, 439–451 (2005)

    Article  CAS  Google Scholar 

  22. 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)

    Article  MathSciNet  CAS  Google Scholar 

  23. Wu, P. Y. et al. A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J. 22, 5241–5250 (2003)

    Article  CAS  Google Scholar 

  24. Song, J., Durrin, L. K., Wilkinson, T. A., Krontiris, T. G. & Chen, Y. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl Acad. Sci. USA 101, 14373–14378 (2004)

    Article  CAS  ADS  Google Scholar 

  25. Hannich, J. T. et al. Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae . J. Biol. Chem. 280, 4102–4110 (2005)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Leverson, J. D. et al. The APC11 RING-H2 finger mediates E2-dependent ubiquitination. Mol. Biol. Cell 11, 2315–2325 (2000)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Strickland, S., Palmer, G. & Massey, V. Determination of dissociation constants and specific rate constants of enzyme–substrate (or protein–ligand) interactions from rapid reaction kinetic data. J. Biol. Chem. 250, 4048–4052 (1975)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. J. Matunis for the original clone containing Nup358/RanBP2 (residues 2,596–2,836), and K. R. Rajashankar and A. Yunus for discussion and for reagents that contributed to this work. Use of the Advanced Photon Source (APS) is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences. Use of the SGX Collaborative Access Team beamline facilities at Sector 31 of the APS was provided by Structural GenomiX, Inc., which constructed and operates the facility. D.R. and C.D.L. were supported in part by a National Institutes of Health grant. C.D.L. acknowledges support from the Rita Allen Foundation.

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Correspondence to Christopher D. Lima.

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Coordinates have been deposited with the Protein Data Bank under accession number 1Z5S. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Table S1

Crystallographic data, phasing, and refinement statistics. (DOC 53 kb)

Supplementary Figure S1

Activities of the Nup358/RanBP2 E3 with RanGAP1 and RanGAP1 (F562A). a) Activity under multiple turnover conditions with RanGAP1 and RanGAP1(F562A). b) Activity under single turnover conditions with RanGAP1 and RanGAP1(F562A). (PDF 61 kb)

Supplementary Figure S2

Biochemical activities of Nup358/RanBP2 with SUMO-2 and SUMO-3. a) For SUMO-2 conjugation. b) For SUMO-3 conjugation. c) Table of rate constants and relative rates for reactions in a and b. (PDF 29 kb)

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Reverter, D., Lima, C. Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex. Nature 435, 687–692 (2005). https://doi.org/10.1038/nature03588

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