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.

Ubiquitin-Dependent Sorting in Endocytosis

“…Epsin favors binding to K63-polyubiquitin, which correlates well with the requirement of multiple Ubs as an internalization signal (Hawryluk et al 2006;Sato et al 2009). Other UBDs are present at sites of CME including ones in CIN85 and Hrs, and others are likely to be uncovered (Soubeyran et al 2002;Stamenova et al 2007;Mayers et al 2013).…”

The ubiquitin landscape at DNA double-strand breaks

“…2 A). The structure reveals that these domains consists of a single 60-Å long -helix (Sato et al, 2009). Interestingly, the inter-UIM region adopts an -helical secondary structure, as predicted by Sims and Cohen (2009).…”

“…The RNF168 MIU domains have a much larger intervening sequence, and although in vitro ubiquitin binding to K63-Ub has been described (Penengo et al, 2006), the exact configuration the linker region between the two UIMs was important for binding K63-linked polyubiquitin. Sato et al, (2009) recently described the mouse RAP80 tandem UIM domain structure bound to K63-linked diubiquitin (Fig. 2 A).…”

“…It serves as a molecular ruler measuring the distance between K63-linked ubiquitin and orients the hydrophobic regions to lie on one side. These findings led to a model of linkage-specific avidity, in which linker region (Komander et al, 2009;Sato et al, 2009). CtIP with BRCA1 and Mre11.…”

Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

“…UIMs are part of a larger class of ubiquitin-binding domains (UBDs) formed from a single α helix that is often found in multiple arrays (Hicke et al 2005;Husnjak and Dikic 2012). Tandem UIMs have been shown to bind K63-linked ubiquitin chains in the mammalian DNA repair protein RAP80 (Sato et al 2009). To assess their function in DA1, the N-terminal region of DA1 containing mutated UIM1 and UIM2 was fused to GST and expressed in Escherichia coli, and conserved Ala and Ser residues, predicted to be in the α-helical domain of the UIMs (Supplemental Fig.…”