NMR analysis of Lys63-linked polyubiquitin recognition by the tandem ubiquitin-interacting motifs of Rap80
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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.
KeywordsUbiquitin Lys63-linked polyubiquitin chains Rap80 Tandem ubiquitin-interacting motifs NMR spectroscopy
We thank Dr. Akio Ojida and Dr. Itaru Hamachi for their help in collecting the ITC data, the NMR Facility of the Yokohama Institute at RIKEN and the High-field Biomacromolecular NMR Core Facility, National Research Program for Genomic Medicine, Taiwan, Republic of China, for collecting the NMR data. This work was supported by grants to M.S. and H.T. from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and the Japan Science and Technology Agency. This research was also supported in part by the Global COE Program “International Center for Integrated Research and Advanced Education in Materials Science” (No. B-09) of MEXT of Japan, administered by the Japan Society for the Promotion of Science.
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