Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80.
RAP80 has a key role in the recruitment of the Abraxas-BRCC36-BRCA1-BARD1 complex to DNA-damage foci for DNA repair through specific recognition of Lys 63-linked polyubiquitinated proteins by its tandem ubiquitin-interacting motifs (UIMs). Here, we report the crystal structure of the RAP80 tandem UIMs (RAP80-UIM1-UIM2) in complex with Lys 63-linked di-ubiquitin ... at 2.2 A resolution. The two UIMs, UIM1 and UIM2, and the alpha-helical inter-UIM region together form a continuous 60 A-long alpha-helix. UIM1 and UIM2 bind to the proximal and distal ubiquitin moieties, respectively. Both UIM1 and UIM2 of RAP80 recognize an Ile 44-centered hydrophobic patch on ubiquitin but neither UIM interacts with the Lys 63-linked isopeptide bond. Our structure suggests that the inter-UIM region forms a 12 A-long alpha-helix that ensures that the UIMs are arranged to enable specific binding of Lys 63-linked di-ubiquitin. This was confirmed by pull-down analyses using RAP80-UIM1-UIM2 mutants of various length inter-UIM regions. Further, we show that the Epsin1 tandem UIM, which has an inter-UIM region similar to that of RAP80-UIM1-UIM2, also selectively binds Lys 63-linked di-ubiquitin.
Mesh Terms:
Amino Acid Motifs, Animals, Carrier Proteins, Cell Cycle Proteins, Crystallography, X-Ray, Lysine, Mice, Models, Molecular, Molecular Sequence Data, Mutant Proteins, Nuclear Proteins, Polyubiquitin, Protein Binding, Protein Conformation, Sequence Alignment, Transcription Factors
Amino Acid Motifs, Animals, Carrier Proteins, Cell Cycle Proteins, Crystallography, X-Ray, Lysine, Mice, Models, Molecular, Molecular Sequence Data, Mutant Proteins, Nuclear Proteins, Polyubiquitin, Protein Binding, Protein Conformation, Sequence Alignment, Transcription Factors
EMBO J.
Date: Aug. 19, 2009
PubMed ID: 19536136
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