Structures of the Human Poly (ADP-Ribose) Glycohydrolase Catalytic Domain Confirm Catalytic Mechanism and Explain Inhibition by ADP-HPD Derivatives

Abstract

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5′-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

Structures and Mechanisms of Enzymes Employed in the Synthesis and Degradation of PARP-Dependent Protein ADP-Ribosylation

“…Human as well as other vertebrate PARGs are composed of three domains: two of these domains, namely macrodomain and the PARG accessory domain, make up the minimal PARG catalytic region, while the third domain, which shows significantly less sequence conservation, is the putative PARG regulatory region (Figure 4) (Dunstan et al, 2012;Patel et al, 2005;Slade et al, 2011;Tucker et al, 2012). The latter regulatory domain is absent in PARGs of the majority of eukaryotes, and it is not important for PAR hydrolysis in vitro (Botta and Jacobson, 2010;Slade et al, 2011).…”

“…Similarly to other macrodomains, the macrodomain in the PARG catalytic region also has a compact globular shape with a central 7-stranded mixed b sheet flanked by five a helices, packed onto both sides of the sheet, with one helix present at the ridge of the sheet (Dunstan et al, 2012;Kim et al, 2012;Slade et al, 2011;Tucker et al, 2012;Wang et al, 2014). In addition, in all canonical macrodomain proteins, including PARG, the ADP-ribose binding sites are typically situated at a crest of the globular domain, with central phosphates typically buried inside a tunnel ( Figure 5).…”

“…As revealed by the co-structural data, the N-ribose 0 (also referred to as adenine ribose) is normally found buried in the active site. In contrast, the proximal N-ribose 00 (nicotinamide ribose) is situated in a crevice lined by overall positive charge, which in turn is proposed to accommodate the negatively charged ADP-ribose phosphate groups ( Figure 5A) (Kim et al, 2012;Tucker et al, 2012).…”

“…This loop bears the highly conserved GGG-X 6-8 -QEE PARG signature sequence, which contains the catalytic residues essential for PAR degradation activity (Patel et al, 2005;Slade et al, 2011;Tucker et al, 2012). The acid-base catalysis of PAR degradation relies on the presence of the PARG catalytic loop, which allows for PAR hydrolysis, which in turn occurs via an oxocarbenium intermediate and is described in detail by Barkauskaite et al (2013a) and Slade et al (2011).…”

“…Furthermore, this PARG-PAR structure also revealed that PARGs preferably bind PAR at the chain termini and primarily act as exo-glycohydrolases (whereby PARG sequentially degrades terminal ADP-ribose units). While binding along the PAR chain and endo-glycohydrolytic cleavage of PAR is structurally possible, it appears to be less efficient (Barkauskaite et al, 2013a;Braun et al, 1994;Lambrecht et al, 2015;Tucker et al, 2012). Such PARG endo-glycohydrolase activity may become significant in vivo at high PAR/ PARG ratios (for example, in the case of an extreme cellular insult), thus releasing free PAR fragments to mediate apoptotic signaling (Andrabi et al, 2006;Barkauskaite et al, 2013a).…”