Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80

Abstract

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.

Figure 1

Overall structure of the RAP80-UIM1-UIM2–K63-Ub₂ complex. (A) Schematic view of mouse RAP80. UIM domain and AIR (Abraxas-interacting region) interact with Lys 63-linked polyubiquitin chains and Abraxas, respectively. (B) Pull-down assays using GST-fused RAP80-UIM1-UIM2 to bind to K63-, K48- or linear Ub₂. (C) The UIMs and inter-UIM region of RAP80-UIM1-UIM2 are coloured grey and red, respectively. The proximal and distal ubiquitin moieties are coloured pink and cyan, respectively. Ile 44 in the both of the ubiquitin moieties is shown as green spheres. Lysine residues and the N-terminal methionine of the proximal ubiquitin are shown as spheres. The K63R mutation in the distal ubiquitin (used to generate K63-Ub₂) is shown by a stick model.

Figure 2

Stereo view of the interface between the ubiquitin moieties and RAP80-UIM1-UIM2. The colouring scheme is the same as in Figure 1. The labels of RAP80, the proximal, and distal ubiquitin moieties are coloured black, red, and blue, respectively. (A) The interface between the proximal ubiquitin and the UIM1. (B) The interface between the distal ubiquitin and the UIM2.

Figure 3

The order of the two UIMs of RAP80-UIM1-UIM2 is not critical for selective binding of K63-Ub₂. (A) Amino-acid sequence alignment of the tandem UIMs of RAP80. The residue numbers of mouse RAP80 are indicated above the sequence alignment. Identical and more than 70% identical residues are highlighted by red background and red characters, respectively. Residues that hydrogen bond to the proximal and distal ubiquitin moieties are marked with pink and cyan circles, respectively. Residues involved in hydrophobic interactions with the proximal and distal ubiquitin moieties are marked with pink and cyan triangles, respectively. Grey and red bars below the alignment correspond to the UIMs and inter-UIM region, respectively. (B) Amino-acid sequence alignment of the UIM1 and UIM2 of the mouse RAP80. The symbols and colouring are the same as in Figure 3A. (C) Amino-acid sequences of the UIM1–UIM1, UIM2–UIM2, and UIM2–UIM1 of RAP80. (D) Pull-down assays using the GST-fused RAP80-UIM1-UIM2 mutants and K63-Ub₂.

Figure 4

The length of the inter-UIM region of RAP80-UIM1-UIM2 is critical for its ability to bind K63-Ub₂. (A) Amino-acid sequences of the inter-UIM region of the RAP80 mutants used for the GST pull-down assay. (B) Schematic view of residue positions in RAP80-UIM1-UIM2. Ala 88 and Ala 113, the centre of the ubiquitin-binding surfaces of the RAP80 UIM1 and UIM2, are coloured pink and cyan, respectively. The putative positions of Ala 113 in the −1, +2, and +4 mutants of RAP80 are coloured red. (C) Pull-down assays using GST-fused RAP80-UIM1-UIM2 mutants and K63-, K48-, or linear Ub₂.

Figure 5

The tandem UIMs of Epsin1 specifically binds to K63-Ub₂. The labels UIM-12, UIM-23, and UIM-13 represent UIM1–UIM2, UIM2–UIM3 and UIM1–UIM3 of Epsin1, respectively. (A) Amino-acid sequence alignment of the tandem UIMs identified to date in the mouse genome. The symbols and colouring are the same as in Figure 3A. RAP80 and Epsin1 are highlighted by yellow background. (B) Pull-down assays using GST-fused RAP80-UIM1-UIM2 and Epsin1 to bind K63-, K48-, or linear Ub₂.