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.
Sato
Yusuke
Y
Structural Biology Laboratory, Life Science Division, Synchrotron Radiation Research Organization and Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.
Yoshikawa
Azusa
A
Mimura
Hisatoshi
H
Yamashita
Masami
M
Yamagata
Atsushi
A
Fukai
Shuya
S
D020816:Amino Acid Motifs; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D050505:Mutant Proteins; D009687:Nuclear Proteins; C543502:Rap80 protein, mouse; D014157:Transcription Factors; C466234:UIMC1 protein, human; D025821:Polyubiquitin; D008239:Lysine