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By WWE Domains Suggests a General Mechanism for Poly (ADP-Ribosyl)

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By WWE Domains Suggests a General Mechanism for Poly (ADP-Ribosyl) Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION Zheng 2009). Protein poly(ADP-ribosyl)ation (PARylation), Recognition of the catalyzed by PAR polymerases (PARPs), also regulates a iso-ADP-ribose moiety in myriad of biological processes, including DNA damage responses, transcriptional regulation, intracellular traffick- poly(ADP-ribose) by WWE ing, energy metabolism, circadian rhythm, and cell sur- vival and cell death programs, among others (Curtin 2005; domains suggests a general Jagtap and Szabo 2005; Kim et al. 2005; Schreiber et al. mechanism for poly 2006; Krishnakumar and Kraus 2010). How PARylation affects so many biological functions remains largely mys- (ADP-ribosyl)ation-dependent terious. In many cases, such as PARylation of histones in ubiquitination transcriptional regulation, PARylation is considered to control activities of the substrate proteins via the nega- Zhizhi Wang,1,2 Gregory A. Michaud,3 tive charges in the PAR polymer (Schreiber et al. 2006; Zhihong Cheng,1 Yue Zhang,3 Thomas R. Hinds,4 Krishnakumar and Kraus 2010). In other cases, PAR poly- 1,5 3 1,6 mers have been implicated as signaling molecules that can Erkang Fan, Feng Cong, and Wenqing Xu induce cell death, especially in the brain (Andrabi et al. 1Department of Biological Structure, 2Biomolecular Structure 2006, 2011). and Design Program, University of Washington, Seattle, Most recently, PARylation has been shown to control Washington 98195, USA; 3Novartis Institutes for Biomedical the polyubiquitination and degradation of axin, a key reg- Research, Cambridge, Massachusetts 02139, USA; 4Department ulator of the Wnt signaling pathway (Huang et al. 2009; of Pharmacology, 5Department of Biochemistry, University Callow et al. 2011; Kang et al. 2011; Zhang et al. 2011). In of Washington, Seattle, Washington 98195, USA 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 Protein poly(ADP-ribosyl)ation and ubiquitination are two PARylation of the substrate for subsequent polyubiqui- key post-translational modifications regulating many bio- tination (Callow et al. 2011; Kang et al. 2011; Zhang et al. logical processes. Through crystallographic and biochem- 2011). The RNF146 WWE domain has been shown to bind ical analysis, we show that the RNF146 WWE domain PAR (Callow et al. 2011; Zhang et al. 2011), and it was recognizes poly(ADP-ribose) (PAR) by interacting with reported that a short PAR-binding motif (PBM) within the iso-ADP-ribose (iso-ADPR), the smallest internal PAR domain retains this binding activity (Andrabi et al. 2011). structural unit containing the characteristic ribose–ribose The PBM was originally found in histones and several glycosidic bond formed during poly(ADP-ribosyl)ation. other proteins (Gagne et al. 2008). However, the PBM iden- tified in RNF146 is not conserved in other WWE domains, The key iso-ADPR-binding residues we identified are so it remains unclear whether the WWE domain repre- highly conserved among WWE domains. Binding assays sents a novel PAR-binding domain. Here we reveal the further demonstrate that PAR binding is a common func- structural basis of the RNF146 WWE domain/iso-ADPR tion for the WWE domain family. Since many WWE do- interaction and, for the first time, define the PAR/iso- main-containing proteins are known E3 ubiquitin ligases, ADPR binding as a bona fide function of the WWE domain our results suggest that protein poly(ADP-ribosyl)ation family. Importantly, the structural coupling of WWE do- may be a general mechanism to target proteins for mains and E3 ligase domains in many WWE domain- ubiquitination. containing proteins suggests a functional coupling of pro- tein PARylation and ubiquitination. Supplemental material is available for this article. Received November 2, 2011; revised version accepted December 19, 2011. Results and Discussion The RNF146 WWE domain recognizes iso-ADPR, but not ADPR Protein ubiquitination regulates diverse biological pro- We sought to clarify the requirement of the entire cesses; however, the mechanism by which proteins are RNF146 WWE domain structure for PAR binding through earmarked for ubiquitination remains incompletely un- structural analysis. PAR polymers display high chemical derstood. Other than phosphorylation, which is a general heterogeneity (in both lengths and branching patterns) mechanism for many cases, hydroxylation of a substrate and are not suitable for quantitative and structural anal- (i.e., HIF1-a) and the binding of small molecules (e.g., the ysis. Thus, we first examined its interaction with ADP- plant hormone auxin) to E3 ligases have been shown to con- ribose (ADPR), the building unit added to PAR during PAR trol protein ubiquitination in sporadic cases (Willems et al. synthesis (Fig. 1A). Isothermal titration calorimetry (ITC) 1999; Min et al. 2002; Bergink and Jentsch 2009; Tan and analysis demonstrated that the RNF146 WWE domain does not interact with ADPR, even at high concentrations [Keywords: poly(ADP-ribosyl)ation; ubiquitination; WWE domain; PARP-1; (>0.1 mM) (Fig. 1C). We then turned to iso-ADPR, which axin; crystal structure] 6 is the smallest PAR structural unit containing the ribose– Corresponding author. ribose glycosidic bond unique to PAR, formed during PAR E-mail [email protected]. Article published online ahead of print. Article and publication date are synthesis by PARPs (Fig. 1A). It remains a major challenge online at http://www.genesdev.org/cgi/doi/10.1101/gad.182618.111. to obtain PAR of a specific length in sufficient quantities GENES & DEVELOPMENT 26:000–000 Ó 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org 1 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Wang et al. Crystal structure of the RNF146 WWE domain in complex with iso-ADPR To understand how the RNF146 WWE domain interacts with iso-ADPR, we determined the crystal structure of the RNF146 WWE domain in complex with iso-ADPR at 1.63 A˚ resolution (Fig. 2A,B; Supplemental Table 1). The WWE domain contains six b strands, forming half of a b barrel, with the other side of this half b barrel covered by an a helix. The high resolution of our structure al- lowed us to define unambiguously that the ribose–ribose linkage in iso-ADPR is an a(1 / 2) glycosidic bond (Fig. 2B). This confirmed that PAR synthesis catalyzed by PARP1 is a(1 / 2)-spe- cific. The adenine ring of iso-ADPR inserts into the pocket formed by the half b barrel and the a helix (Fig. 2A). The two ribose–phosphate moieties on both sides of iso-ADPR sit on the edge of the half b barrel, which is highly positively charged (Fig. 2C). Both sides of iso-ADPR, especially the two sepa- rated phosphate groups, are involved in Figure 1. The interaction between the RNF146 WWE domain and PAR, through the recognition of iso-ADPR. (A) Structures of PAR and its structural units. ADPR is the unit extensive interactions with WWE do- within the blue frame, and iso-ADPR is within the red frame. (B) Procedural outline of iso- main residues (Fig. 2D), providing an ADPR in vitro biosynthesis and purification. (C) ITC analysis showed the binding of the explanation for why the RNF146 WWE RNF146 WWE domain with iso-ADPR (Kd ; 370 nM), but not with ADPR. domain specifically binds to iso-ADPR and thus to PAR, but not ADPR, which has the two phosphate groups on the for biochemical analysis, and iso-ADPR is not commer- same side (Fig. 1A). Compared with a previously deter- cially available, nor has it been used previously in struc- mined nuclear magnetic resonance (NMR) structure of an tural and biochemical studies. We therefore developed a unliganded RNF146 WWE domain (Protein Data Bank protocol to biosynthetically generate iso-ADPR (Fig. 1B). [PDB] code 1UJR), it appears that iso-ADPR binding in- We synthesized PAR using histone PARylation by PARP1, duces significant conformational changes in the WWE following previously published protocols (Kiehlbauch et al. domain, particularly in the C-terminal tail region (resi- 1993; Fahrer et al. 2007). After removing small molecules dues 169–183), which folds back to support the distal by size exclusion chromatography (SEC), we digested PAR ribose–phosphate groups of iso-ADPR (Supplemental Fig. polymers with a phosphodiesterase to generate iso-ADPR. 3). The mode of binding displayed in our crystal structure, Finally, we purified iso-ADPR by ion exchange and a sec- in which the two phosphate groups lie on an open surface ond SEC step to remove all remaining large molecules. of the RNF146 WWE domain, should allow internal iso- The purity and identity of purified iso-ADPR were con- ADPR units in PAR polymers to bind the WWE domain in firmed by reverse-phase high-performance liquid chroma- the same manner. tography (RP-HPLC) and mass spectrometry. ITC analysis demonstrated that, in contrast to the poor interaction with Mutagenesis analysis of the RNF146 WWE domain ADPR, the RNF146 WWE domain interacted with iso- ADPR avidly, with a dissociation constant of 0.37 mM Seven RNF146 WWE domain surface residues are involved (Fig. 1C). Selectivity of the RNF146 WWE domain for iso- in iso-ADPR binding (Fig. 2D). Among them, the Tyr 107 ADPR was also demonstrated by its comigration with phenol group stacks on the side of the iso-ADPR adenine iso-ADPR, but not ADPR, in SEC (Supplemental Fig. 2). ring within the pocket, and Gln 153 near the bottom of Thus, the RNF146 WWE domain interacts with the PAR the adenine-binding pocket forms two hydrogen bonds polymer, but not mono-ADPR. This is likely to be impor- with the adenine ring and appears to confer binding spec- tant for RNF146 function, since ADP-ribosylation and ificity as well as binding affinity.
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