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The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type

Nature Structural & Molecular Biology volume 11, pages 984–991 (2004)Cite this article

Abstract

Post-translational modification with the ubiquitin-related protein SUMO1 requires the E1 enzyme Aos1–Uba2 and the E2 enzyme Ubc9. Distinct E3 ligases strongly enhance modification of specific targets. The SUMO E3 ligase RanBP2 (also known as Nup358) has no obvious similarity to RING- or HECT-type enzymes. Here we show that RanBP2's 30-kDa catalytic fragment is a largely unstructured protein. Despite two distinct but partially overlapping 79-residue catalytic domains, one of which is sufficient for maximal activity, RanBP2 binds to Ubc9 in a 1:1 stoichiometry. The identification of nine RanBP2 and three Ubc9 side chains that are important for RanBP2-dependent SUMOylation indicates largely hydrophobic interactions. These properties distinguish RanBP2 from all other known E3 ligases, and we speculate that RanBP2 exerts its catalytic effect by altering Ubc9's properties rather than by mediating target interactions.

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Figure 1: Automodification and E3 ligase activity of RanBP2 deletion fragments.
Figure 2: Ubc9 interacts with IR1 and more stably with IR1+M.
Figure 3: IR1+M catalyzes Sp100 SUMOylation efficiently.
Figure 4: Shorter RanBP2 fragments have reduced target specificity.
Figure 5: Cysteine mutants of IR1 have residual ligase activity.
Figure 6: RanBP2 mutants defective for activity and Ubc9 binding.
Figure 7: Ubc9 mutants defective in RanBP2-dependent modification.
Figure 8: IR1+M and M+IR2 are both highly active in Sp100 SUMOylation.
Figure 9: The largely unstructured BP2ΔFG binds Ubc9 in a 1:1 stoichiometry.
Figure 10

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Acknowledgements

Our special thanks go to A. Klanner, J. Vordemann, P. van Dijk and E. Stieger for excellent technical assistance, to members of the lab and L. Hengst for sharing reagents and advice. We acknowledge L. Moroder and E. Weyher (Max Planck Institute for Biochemistry) for CD spectroscopy, and J. Velgaard Olsen (University of Southern Denmark) for mass spectrometry analysis. We thank J. Seeler, A. Dejean, R. Grosschedl, G. Suske, M. Oren and C. Lima for providing clones for GST-Sp100, GST-HDAC4, His-Lef-1, GST-PIAS1, GST-p53, and His-Ubc9, respectively. Funding was provided by BioFUTURE grant 0311869 to F.M.; P.K. was funded through grant NWO-MW901-02-223.

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Authors and Affiliations

  1. Max-Planck Institute for Biochemistry, Am Klopferspitz 18, Martinsried, 82152, Germany

    Andrea Pichler & Frauke Melchior

  2. Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands

    Puck Knipscheer & Titia K Sixma

  3. Institute for Molecular Embryology and Genetics, Kumamoto University, Japan

    Hisato Saitoh

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  1. Andrea Pichler
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Correspondence to Frauke Melchior.

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Supplementary information

Supplementary Fig. 1

N85Q Ubc9 in thioester bond formation and sumoylation. (PDF 72 kb)

Supplementary Fig. 2

Molecular mass of the RanBP2–Ubc9 complex. (PDF 57 kb)

Supplementary Fig. 3

Activity and binding for different RanBP2 fragments. (PDF 40 kb)

Supplementary Fig. 4

Sequence alignment of RanBP2's E3 ligase domain. (PDF 46 kb)

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Pichler, A., Knipscheer, P., Saitoh, H. et al. The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type. Nat Struct Mol Biol 11, 984–991 (2004). https://doi.org/10.1038/nsmb834

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