NMR analysis of Lys63-linked polyubiquitin recognition by the tandem ubiquitin-interacting motifs of Rap80

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NMR analysis of Lys63-linked polyubiquitin recognition by the tandem ubiquitin-interacting motifs of Rap80


Abstract Ubiquitin is a post-translational modifier that is involved in cellular functions through its covalent attachment to target proteins. Ubiquitin can also be conjugated to itself at seven lysine residues and at its amino terminus to form eight linkage-specific polyubiquitin chains for individual cellular processes. The Lys63-linked polyubiquitin chain is recognized by tandem ubiquitin-interacting motifs (tUIMs) of Rap80 for the regulation of DNA repair. To understand the recognition mechanism between the Lys63-linked diubiquitin (K63-Ub2) and the tUIMs in solution, we determined the solution structure of the K63-Ub2:tUIMs complex by using NOE restraints and RDC data derived from NMR spectroscopy. The structure showed that the tUIMs adopts a nearly straight and single continuous α-helix, and the two ubiquitin units of the K63-Ub2 separately bind to each UIM motif. The interfaces are formed between Ile44-centered patches of the two ubiquitin units and the hydrophobic residues of the tUIMs. We also showed that the linker region between the two UIM motifs possesses a random-coil conformation in the free state, but undergoes the coil-to-helix transition upon complex formation, which simultaneously fixes the relative position of ubiquitin subunits. These data suggest that the relative position of ubiquitin subunits in the K63-Ub2:tUIMs complex is essential for linkage-specific binding of Rap80 tUIMs.

• Content Type Journal Article
• Category Article
• Pages 1-12
• DOI 10.1007/s10858-012-9614-9
• Authors
  • Naotaka Sekiyama, Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto, 615-8510 Japan
  • JunGoo Jee, Center for Priority Areas, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo, 192-0397 Japan
  • Shin Isogai, Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto, 615-8510 Japan
  • Ken-ichi Akagi, Section of Laboratory Equipments, National Institute of Biomedical Innovation, 7-6-8, Asagi, Saito, Ibaraki, Osaka, 567-0085 Japan
  • Tai-huang Huang, Division of Structural Biology, Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, 11529 Taiwan
  • Mariko Ariyoshi, Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto, 615-8510 Japan
  • Hidehito Tochio, Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto, 615-8510 Japan
  • Masahiro Shirakawa, Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto, 615-8510 Japan

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR