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Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation

Nature Structural & Molecular Biology volume 13, pages 915–920 (2006)Cite this article

Abstract

Lys63-linked polyubiquitin chains participate in nonproteolytic signaling pathways, including regulation of DNA damage tolerance and NF-κB activation. E2 enzymes bound to ubiquitin E2 variants (UEV) are vital in these pathways, synthesizing Lys63-linked polyubiquitin chains, but how these complexes achieve specificity for a particular lysine linkage has been unclear. We have determined the crystal structure of an Mms2–Ubc13-ubiquitin (UEV–E2-Ub) covalent intermediate with donor ubiquitin linked to the active site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Mms2–Ubc13-Ub complex to the acceptor-binding site of Mms2–Ubc13 in an adjacent complex allows us to visualize at atomic resolution the molecular determinants of acceptor-ubiquitin binding. The structure reveals the key role of Mms2 in allowing selective insertion of Lys63 into the Ubc13 active site and suggests a molecular model for polyubiquitin chain elongation.

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Figure 1: The structure of the Mms2–Ubc13-Ub complex.
Figure 2: Molecular details of linkage selectivity.
Figure 3: Roles of Mms2 Ser27 and Mms2 Thr44 in acceptor-ubiquitin binding.
Figure 4: Disrupting key interactions near Lys63 of the acceptor ubiquitin elicits moderate catalytic defects (Coomassie-stained gel).
Figure 5: Model for Lys63-linked polyubiquitin chain formation.

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References

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

    Article  CAS  Google Scholar 

  2. Pickart, C.M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610–616 (2004).

    Article  CAS  Google Scholar 

  3. Sun, L. & Chen, Z.J. The novel functions of ubiquitination in signaling. Curr. Opin. Cell Biol. 16, 119–126 (2004).

    Article  CAS  Google Scholar 

  4. Ulrich, H.D. Degradation or maintenance: actions of the ubiquitin system on eukaryotic chromatin. Eukaryot. Cell 1, 1–10 (2002).

    Article  CAS  Google Scholar 

  5. Pickart, C.M. & Cohen, R.E. Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187 (2004).

    Article  CAS  Google Scholar 

  6. Hicke, L. & Dunn, R. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 19, 141–172 (2003).

    Article  CAS  Google Scholar 

  7. Pickart, C.M. & Eddins, M.J. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta 1695, 55–72 (2004).

    Article  CAS  Google Scholar 

  8. Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).

    Article  CAS  Google Scholar 

  9. Hofmann, R.M. & Pickart, C.M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999).

    Article  CAS  Google Scholar 

  10. VanDemark, A.P., Hofmann, R.M., Tsui, C., Pickart, C.M. & Wolberger, C. Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Cell 105, 711–720 (2001).

    Article  CAS  Google Scholar 

  11. Moraes, T.F. et al. Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13. Nat. Struct. Biol. 8, 669–673 (2001).

    Article  CAS  Google Scholar 

  12. Andersen, P.L. et al. Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J. Cell Biol. 170, 745–755 (2005).

    Article  CAS  Google Scholar 

  13. Ulrich, H.D. The RAD6 pathway: control of DNA damage bypass and mutagenesis by ubiquitin and SUMO. ChemBioChem 6, 1735–1743 (2005).

    Article  CAS  Google Scholar 

  14. Chen, Z.J. Ubiquitin signalling in the NF-kappaB pathway. Nat. Cell Biol. 7, 758–765 (2005).

    Article  CAS  Google Scholar 

  15. Zhou, H. et al. Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO. Nature 427, 167–171 (2004).

    Article  CAS  Google Scholar 

  16. 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  CAS  Google Scholar 

  17. McKenna, S. et al. Energetics and specificity of interactions within UbUevUbc13 human ubiquitin conjugation complexes. Biochemistry 42, 7922–7930 (2003).

    Article  CAS  Google Scholar 

  18. McKenna, S. et al. An NMR-based model of the ubiquitin-bound human ubiquitin conjugation complex Mms2Ubc13. The structural basis for lysine 63 chain catalysis. J. Biol. Chem. 278, 13151–13158 (2003).

    Article  CAS  Google Scholar 

  19. Miura, T., Klaus, W., Gsell, B., Miyamoto, C. & Senn, H. Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution. J. Mol. Biol. 290, 213–228 (1999).

    Article  CAS  Google Scholar 

  20. Spyracopoulos, L., Lewis, M.J. & Saltibus, L.F. Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound states. Biochemistry 44, 8770–8781 (2005).

    Article  CAS  Google Scholar 

  21. Lewis, M.J., Saltibus, L.F., Hau, D.D., Xiao, W. & Spyracopoulos, L. Structural basis for non-covalent interaction between ubiquitin and the ubiquitin conjugating enzyme variant human MMS2. J. Biomol. NMR 34, 89–100 (2006).

    Article  CAS  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. Tsui, C., Raguraj, A. & Pickart, C.M. Ubiquitin binding site of the ubiquitin E2 variant (UEV) protein Mms2 is required for DNA damage tolerance in the yeast RAD6 pathway. J. Biol. Chem. 280, 19829–19835 (2005).

    Article  CAS  Google Scholar 

  24. Piotrowski, J. et al. Inhibition of the 26 S proteasome by polyubiquitin chains synthesized to have defined lengths. J. Biol. Chem. 272, 23712–23721 (1997).

    Article  CAS  Google Scholar 

  25. 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 

  26. Reverter, D. & Lima, C.D. Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex. Nature 435, 687–692 (2005).

    Article  CAS  Google Scholar 

  27. Raasi, S. & Pickart, C.M. Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains. J. Biol. Chem. 278, 8951–8959 (2003).

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  30. Kissinger, C.R., Gehlhaar, D.K. & Fogel, D.B. Rapid automated molecular replacement by evolutionary search. Acta Crystallogr. D Biol. Crystallogr. 55, 484–491 (1999).

    Article  CAS  Google Scholar 

  31. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47, 110–119 (1991).

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Bianchet, S. Gabelli and S. Bouyain for crystallographic assistance, B. Schulman, R. Cohen, B. Garcia-Moreno and C. Fitch for advice and discussions, and C. Ralston of Advanced Light Source beamline 8.2.2 for technical assistance. This work was supported by the Howard Hughes Medical Institute (C.W.) and US National Institutes of Health grant GM60372 (C.M.P.).

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  1. Kamila M Gomez

    Present address: Claflin University, Orangeburg, South Carolina, 29115, USA

  2. Cecile M Pickart: Deceased.

Authors and Affiliations

  1. Department of Biophysics and Biophysical Chemistry and the Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Michael J Eddins, Kamila M Gomez & Cynthia Wolberger

  2. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, 21205, Maryland, USA

    Candice M Carlile & Cecile M Pickart

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  1. Michael J Eddins
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Correspondence to Cynthia Wolberger.

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Supplementary information

Supplementary Fig. 1

SDS gel showing the ternary complex used for crystallization and the contents of the dissolved crystal. (PDF 71 kb)

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Eddins, M., Carlile, C., Gomez, K. et al. Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nat Struct Mol Biol 13, 915–920 (2006). https://doi.org/10.1038/nsmb1148

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