Recognition of the iso-ADP-ribose moiety in poly(ADP-ribose) by WWE domains suggests a general mechanism for poly(ADP-ribosyl)ation-dependent ubiquitination

Abstract

Protein poly(ADP-ribosyl)ation and ubiquitination are two key post-translational modifications regulating many biological processes. Through crystallographic and biochemical analysis, we show that the RNF146 WWE domain recognizes poly(ADP-ribose) (PAR) by interacting with iso-ADP-ribose (iso-ADPR), the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The key iso-ADPR-binding residues we identified are highly conserved among WWE domains. Binding assays further demonstrate that PAR binding is a common function for the WWE domain family. Since many WWE domain-containing proteins are known E3 ubiquitin ligases, our results suggest that protein poly(ADP-ribosyl)ation may be a general mechanism to target proteins for ubiquitination.Supplemental material is available for this article.Received November 2, 2011; revised version accepted December 19, 2011. Protein ubiquitination regulates diverse biological processes; however, the mechanism by which proteins are earmarked for ubiquitination remains incompletely understood. Other than phosphorylation, which is a general mechanism for many cases, hydroxylation of a substrate (i.e., HIF1-a) and the binding of small molecules (e.g., the plant hormone auxin) to E3 ligases have been shown to control protein ubiquitination in sporadic cases (Willems et al. 1999;Min et al. 2002;Bergink and Most recently, PARylation has been shown to control the polyubiquitination and degradation of axin, a key regulator of the Wnt signaling pathway (Huang et al. 2009;Callow et al. 2011;Kang et al. 2011;Zhang et al. 2011). In all of these reports, RNF146 (aka Iduna), which contains a WWE domain and a RING domain (Supplemental Fig. 1), is the only known E3 ubiquitin ligase to date that requires PARylation of the substrate for subsequent polyubiquitination (Callow et al. 2011;Kang et al. 2011;Zhang et al. 2011). The RNF146 WWE domain has been shown to bind PAR (Callow et al. 2011;Zhang et al. 2011), and it was reported that a short PAR-binding motif (PBM) within the domain retains this binding activity ). The PBM was originally found in histones and several other proteins (Gagne et al. 2008). However, the PBM identified in RNF146 is not conserved in other WWE domains, so it remains unclear whether the WWE domain represents a novel PAR-binding domain. Here we reveal the structural basis of the RNF146 WWE domain/iso-ADPR interaction and, for the first time, define the PAR/iso-ADPR binding as a bona fide function of the WWE domain family. Importantly, the structural coupling of WWE domains and E3 ligase domains in many WWE domaincontaining proteins suggests a functional coupling of protein PARylation and ubiquitination. Results and DiscussionThe RNF146 WWE domain recognizes iso-ADPR, but not ADPR We sought to clarify the requirement of the entire RNF146 WWE domain structure for PAR binding through structural analysis. PAR polymers display high chemical heterogeneity (in both lengths and branching patterns) and ar...

The oligonucleotide/oligosaccharide-binding fold motif is a poly(ADP-ribose)-binding domain that mediates DNA damage response

“…S2A, Right). The affinity is similar to that between iso-ADPR and the WWE motif of RNF146, a known iso-ADPR-binding domain (27) (Fig. S2C).…”

PARP12, an Interferon-stimulated Gene Involved in the Control of Protein Translation and Inflammation

“…A possible link between ADP-ribosylation and ubiquitination has been suggested by the shared presence of WWE domains among several enzymes catalyzing these two post-translational modifications (57). The best characterized WWE domain (from the E3 ubiquitin ligase RNF146) has been shown to bind to poly(ADP-ribose) through recognition of the iso-ADP-ribose moiety only found in poly-, but not mono-ADP-ribosyl modified proteins (58). However, other WWE domains display weak to no affinity for iso-ADP-ribose, suggesting that other members of the ubiquitin ligase family may interact with mono-ADP-ribose and possibly mediate proteasome-dependent degradation of monoribosylated substrates.…”

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

“…Using isothermal titration calorimetry (ITC) assays, we measured the affinity between PAR and these FHA/ BRCT domains (Fig. 1C), which is in the physiologically relevant range and is similar to that between PAR and other PAR-binding domains (Karras et al 2005;Wang et al 2012). Since PAR is the ADP-ribose polymer with mixed length and contains both linear and branched forms, the affinity between the PAR-binding domain and PAR could not be accurately measured.…”

“…Purified PAR was fractionated according to chain length by anion exchange high-pressure liquid chromatography (HPLC) protocol. Iso-ADP-ribose was generated The FHA and BRCT domains bind poly(ADP-ribose) and purified in vitro according to the procedure by Wang et al (2012). Briefly, the purified PAR was digested by 50 U of snake venom phosphodiesterase (Worthington) with 15 mM MgCl 2 overnight at room temperature.…”

“…Recently, several PAR-binding proteins have been identified as the ''readers'' to recognize PAR signals (Karras et al 2005;Ahel et al 2008;Wang et al 2012), suggesting that PAR is likely to function as recruiting signals to induce DNA damage response factors to DNA damage sites. To identify other PAR-binding modules, we examined both the Forkhead-associated (FHA) domain and the BRCA1 C-terminal (BRCT) domain.…”