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

Julie A Tucker,Martin Watson,Caroline Truman,Stephen T Durant,Geoff Holdgate,Giles Hassall,J Willem M Nissink,Mark Mcalister,Claire Brassington,Neil Bennett,Roland Seifert

doi:10.1371/journal.pone.0050889

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.

Highlights

Single-strand breaks (SSBs) are the most frequent type of DNA lesion occurring in prokaryotic and eukaryotic cells
In order to define a minimal catalytic domain construct suitable for structural studies, we created a consensus disorder prediction based on the results of the automated disorder prediction software servers RONN [30], DisEMBL [31] and PrDOS [32] for the human PARG (hPARG) sequence (UNIProt ID: Q86W56)
Our structures confirm the conservation of the overall fold amongst mammalian PAR glycohydrolase (PARG) catalytic domains, as exemplified recently in the structures of rat and mouse PARG ([25] and Protein Data Bank (PDB) ID: 4FC2), whilst highlighting important similarities and differences between the bacterial and protozoal PARG structures [23,24] and those of mammalian PARGs

Summary

Introduction

Single-strand breaks (SSBs) are the most frequent type of DNA lesion occurring in prokaryotic and eukaryotic cells. They commonly arise from direct attack of deoxyribose by intracellular reactive metabolites, as abortive intermediates of topoisomerase 1 activity, or as intermediates occurring as a result of base excision repair (BER) acting to resolve lesions induced by genotoxins such as DNA alkylating and methylating agents [1]. PARP-1 rapidly binds to and is activated by DNA single- and double-strand breaks, resulting in covalent modification of itself and other target proteins with long chains of PAR. PAR polymers average one branch every 20–50 ADP-ribose (ADPR) units. This polymerisation triggers local chromatin relaxation and recruitment of DNA repair factors. The presence of high levels of PAR in cells is, transient because the polymer is rapidly degraded by PAR glycohydrolase (PARG), the only enzyme known to catabolise PAR following DNA damage [2]

Methods

Results

Conclusion