title: 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-Ub(2)) and the tUIMs in solution, we determined the solution structure of the K63-Ub(2):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-Ub(2) 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-Ub(2):tUIMs complex is essential for linkage-specific binding of Rap80 tUIMs.
authors.affiliation: Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
authors.forename: Naotaka
authors.initials: N
authors.lastname: Sekiyama
authors.forename: Jungoo
authors.initials: J
authors.lastname: Jee
authors.forename: Shin
authors.initials: S
authors.lastname: Isogai
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Akagi
authors.forename: Tai-Huang
authors.initials: TH
authors.lastname: Huang
authors.forename: Mariko
authors.initials: M
authors.lastname: Ariyoshi
authors.forename: Hidehito
authors.initials: H
authors.lastname: Tochio
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shirakawa
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D025821:Polyubiquitin / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D009687:Nuclear Proteins; C466234:UIMC1 protein, human; D025821:Polyubiquitin; D008239:Lysine