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.

Figure 1. Crystal structure of a PARG–PAR complex.

(a) Stereoview of the solvent-accessible surface of PARG in grey with the bound PAR at the active-site surface shown in atom-coloured sticks. The 2F_oF_c electron density corresponding to the ordered region of PAR is shown in a blue mesh (contour level 1 sigma). (b) Stereoview of the PARG active site. Residues involved in direct contacts with the PAR ligand are shown in atom-coloured sticks. The mutated Glu256 is shown with green rather than with light blue carbons. Hydrogen bonds between ligand and protein or structural waters are indicated by dotted lines.

Figure 2. PARG mutagenesis and PAR termini protection by bactPARG confirms the PARG–PAR model.

(a) Activity of the T. thermophila PARG WT and mutants and the corresponding human PARG mutants. Error bars represent s.d. (n=3) (*P<0.05; **P<0.01; ***P<0.001) obtained using paired t-test. (b) Inhibition of glycohydrolase activity for both human and T. thermophila PARGs using PARP1-generated PAR substrate with bactPARG Glu115Gln. (c) Distribution of exo- and endo-glycohydrolase products obtained after treatment with wt and mutant human and T. termophila PARGs, as determined by LC/MS (see also Supplementary Fig. S6).

Figure 3. Model of the PARG–PAR complex in endo-glycohydrolase mode.

(a) Solvent-accessible surface of PARG in grey (the position of Phe398 is shown in blue) with a PAR₃ modelled in an endo-glycohydrolase position. Carbons belonging to the three individual ADP-ribose units are coloured distinctly. (b) Stereoview of an overlay of the modelled endo-glycohydrolase PAR₃ (colour coded as in panel a) with the observed exo-glycohydrolase crystal structure. Residues implicated in steric hindrance with the additional N+1 unit are shown in VDW spheres (Phe398 and Leu226). The hydrogen network between the N adenosine and the protein/structural water observed for the exo-glycohydrolase binding mode is shown in dotted lines.

Figure 4. A mechanism for poly-ADP-ribose hydrolysis.

(a) A detailed mechanism for PARG and MacroD1 based on the PARG-PARG structure. R1=pADPr for PARG, glutamate for MacroD1 and R2=ADP. (b) PARG is unable to hydrolyse the terminal ADP-ribose–PARP1 bond. MacroD1 and human PARG activities on radioactively labelled poly(ADP-ribosyl)ated PARP1. MacroD1 WT and the catalytic mutant, G270E, as well as human PARG E756Q mutant, are unable to process PAR. (c) MacroD1 WT removes the terminal ADP-ribose group attached to mono(ADP-ribosyl)ated PARP1 E988Q substrate. In contrast, the MacroD1 catalytic mutant and human PARG do not exhibit the mono(ADP-ribosyl) hydrolase activity.