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

Naotaka Sekiyama, Jungoo Jee, Shin Isogai, Ken Ichi Akagi, Tai Huang Huang, Mariko Ariyoshi, Hidehito Tochio*, Masahiro Shirakawa

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)


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 ahelix, 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.

Original languageEnglish
Pages (from-to)339-350
Number of pages12
JournalJournal of Biomolecular NMR
Issue number4
Publication statusPublished - 2012 Apr
Externally publishedYes


  • Lys63-linked polyubiquitin chains
  • NMR spectroscopy
  • Rap80
  • Tandem ubiquitin-interacting motifs
  • Ubiquitin

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy


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