Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

Abstract

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.

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

“…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).…”

“…The accessory domain (which is part of the PARG catalytic region) has only limited interaction with PAR, but it structurally supports the PARG catalytic loop and may have an important role in regulation of PARG catalytic activity (Barkauskaite et al, 2013a). 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).…”

“…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).…”

“…Protein ADP-Ribosylation Reversal Both PAR and MAR modifications get turned over by the timely action of ADP-ribosyl hydrolases. Interestingly, the main hydrolases involved in this process, namely PAR glycohydrolase (PARG), MacroD1, MacroD2, and terminal ADP-ribose glycohydrolase 1 (TARG1/C6orf130), utilize a macrodomain fold, which not only has evolved to bind the ADP-ribose metabolites, but also has an ADP-ribosyl hydrolase activity (Figure 4) (Barkauskaite et al, 2013a;Jankevicius et al, 2013;Rosenthal et al, 2013;Sharifi et al, 2013;Slade et al, 2011).…”

Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

“…It utilizes NAD (NAD ϩ ) to form PAR at the DNA lesions or breaks, thereby posttranslationally modifying itself and other proteins of interest. Importantly, PAR formation is highly dynamic, because shortly after being synthesized, it is rapidly hydrolyzed by PARP's catabolic counterparts, PAR glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3) (5)(6)(7)(8). Neither enzyme is able to remove the last ADP-ribose moiety from acceptor proteins; macrodomain-containing proteins, such as MacroD1 and MacroD2, fulfill this task, leaving behind an unmodified amino acid that is readily available for the next round of poly-(ADP-ribosyl)ation (PARylation) (9,10).…”