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Structural basis of sugar-recognizing ubiquitin ligase

Nature Structural & Molecular Biology volume 11, pages 365–370 (2004)Cite this article

Abstract

SCFFbs1 is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel β-sandwich. The structure of the SBD–chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.

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Figure 1: Tertiary structure of SBD in Fbs1.
Figure 2: Structure of SBD in complex with chitobiose.
Figure 3: Residues required for interaction of Fbs1 and glycoproteins with high-mannose oligosaccharides.
Figure 4: Identification of the carbohydrate-binding sites of Fbs1 by NMR spectroscopy.

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Acknowledgements

We thank Y. Wada for her help in the preparation of isotopically labeled recombinant proteins and E. Adachi for expert technical assistance. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (K.T. and K.K.), Japan Society for the Promotion of Science and Mizutani Foundation for Glycoscience (K.K.), and in part by the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sport, Science and Technology of Japan (T.T.).

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Authors and Affiliations

  1. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Kawaguchi, 332-0012, Saitama, Japan

    Tsunehiro Mizushima

  2. Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, 113-8613, Japan

    Tsunehiro Mizushima, Yukiko Yoshida, Tomoki Chiba & Keiji Tanaka

  3. Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan

    Takeshi Hirao, Yoshiki Yamaguchi & Koichi Kato

  4. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, 332-0012, Saitama, Japan

    Yukiko Yoshida, Kazuhiro Iwai, Yoshiki Yamaguchi & Koichi Kato

  5. Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, 565-0871, Osaka, Japan

    Soo Jae Lee & Tomitake Tsukihara

  6. Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, Osaka, 545-8585, Japan

    Kazuhiro Iwai

  7. Genomic Sciences Center, RIKEN Yokohama Institute, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan

    Koichi Kato

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Correspondence to Keiji Tanaka.

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Mizushima, T., Hirao, T., Yoshida, Y. et al. Structural basis of sugar-recognizing ubiquitin ligase. Nat Struct Mol Biol 11, 365–370 (2004). https://doi.org/10.1038/nsmb732

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