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Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting

Nature Structural & Molecular Biology volume 13, pages 272–277 (2006)Cite this article

Abstract

Hrs has an essential role in sorting of monoubiquitinated receptors to multivesicular bodies for lysosomal degradation, through recognition of ubiquitinated receptors by its ubiquitin-interacting motif (UIM). Here, we present the structure of a complex of Hrs-UIM and ubiquitin at 1.7-Å resolution. Hrs-UIM forms a single α-helix, which binds two ubiquitin molecules, one on either side. These two ubiquitin molecules are related by pseudo two-fold screw symmetry along the helical axis of the UIM, corresponding to a shift by two residues on the UIM helix. Both ubiquitin molecules interact with the UIM in the same manner, using the Ile44 surface, with equal binding affinities. Mutational experiments show that both binding sites of Hrs-UIM are required for efficient degradative protein sorting. Hrs-UIM belongs to a new subclass of double-sided UIMs, in contrast to its yeast homolog Vps27p, which has two tandem single-sided UIMs.

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Figure 1: Structure of the Hrs-UIM–ubiquitin complex.
Figure 2: Interaction modes of Hrs-UIM with two ubiquitin molecules.
Figure 3: SPR biosensor data from the UIM-ubiquitin interaction, with fitted curves shown.
Figure 4: Assay of EGF degradation.
Figure 5: Putative double-sided UIMs.

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Acknowledgements

This work was supported in part by the Protein 3000 project and by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, as well as by the Norwegian Cancer Society, the Research Council of Norway and the Novo-Nordisk Foundation.

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Authors and Affiliations

  1. Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization, Tsukuba, 305–0801, Ibaraki, Japan

    Satoshi Hirano, Masato Kawasaki, Hideaki Ura, Ryuichi Kato & Soichi Wakatsuki

  2. Department of Biochemistry, Faculty Division of the Norwegian Radium Hospital, University of Oslo, Montebello, Oslo, N-0310, Norway

    Camilla Raiborg & Harald Stenmark

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  1. Satoshi Hirano
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  2. Masato Kawasaki
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Correspondence to Soichi Wakatsuki.

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Supplementary information

Supplementary Table 1

Sequences of synthesized DNA fragments for GST-UIM (PDF 9 kb)

Supplementary Table 2

Sequences of QuikChange primers for UIM mutants (PDF 14 kb)

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Hirano, S., Kawasaki, M., Ura, H. et al. Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting. Nat Struct Mol Biol 13, 272–277 (2006). https://doi.org/10.1038/nsmb1051

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