Crystallographic and biochemical analysis of the mouse poly(ADP-ribose) glycohydrolase

Abstract

Protein poly(ADP-ribosyl)ation (PARylation) regulates a number of important cellular processes. Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for hydrolyzing the poly(ADP-ribose) (PAR) polymer in vivo. Here we report crystal structures of the mouse PARG (mPARG) catalytic domain, its complexes with ADP-ribose (ADPr) and a PARG inhibitor ADP-HPD, as well as four PARG catalytic residues mutants. With these structures and biochemical analysis of 20 mPARG mutants, we provide a structural basis for understanding how the PAR polymer is recognized and hydrolyzed by mPARG. The structures and activity complementation experiment also suggest how the N-terminal flexible peptide preceding the PARG catalytic domain may regulate the enzymatic activity of PARG. This study contributes to our understanding of PARG catalytic and regulatory mechanisms as well as the rational design of PARG inhibitors.

Figure 1. Mouse PARG catalytic domain apo- and ligand bound structures.

(a) Overall structure of apo- mPARG(439–959). The protein is shown in rainbow and the N terminal MTS containing loop is in black. The cleft right in the middle is the active site. (b) ADPr bound mPARG structure. mPARG is shown in light orange and the ADPr is in cyan. Stereoview of key interactions involved in ADPr binding with the mPARG catalytic domain are shown in black dash lines. The key binding residues are highlighted in green sticks. (c) ADP-HPD bound mPARG structure. mPARG is shown in gray and the ADPr is in green. Stereoview of key interactions involved in ADP-HPD binding with the mPARG catalytic domain are shown in black dash lines. The key binding residues are highlighted in pink sticks. Aromatic rings of Tyr788 and Phe895 form perpendicular and parallel π stacking interactions with the adenine ring of ADPr or ADP-HPD, respectively. Tyr785 and Glu720 both form hydrogen bonds with the NH₂ group of the adenine ring. Thr718 and Ile719 are in close contact with the N1 of the adenine ring. In addition, Asn862 forms a hydrogen bond to 2′-OH of the adenine-linked ribose. Tyr788 also forms a hydrogen bond with one of the phosphates. (d) Superposition of unliganded mPARG (blue) and ADP-HPD bound mPARG (grey) structures.Three key loops are highlighted in red in ADP-HPD bound structure. Loop 2 undergoes conformational change to tightly pack the ADP-HPD. Both side chains of Phe868 and Phe895 (highlighted in grey sticks) rotate to strongly interact with ADP-HPD. (e) Superposition of ADP-HPD bound vertebrate PARG catalytic domains. ADP-HPD bound mPARG is in grey, ADP-HPD bound rPARG (PDB: 3UEL) is in wheat and ADP-HPD bound hPARG (PDB: 4B1J) is in magenta. ADP-HPD is showed in cyan stick.

Figure 2. Mutagenesis analysis of mPARG active site residues.

(a) The active site of ADPr bound mPARG structure is shown in light orange. The ligand PAR is modeled in based on superposition of the ADPr bound mPARG structure with PAR bound T. thermophila PARG structure (PDB: 4L2H). PAR is shown in cyan stick, and the residues we designed for mutagenesis study are shown in pink stick. (b) 1 min and 1 h PARG TLC assay for wt mPARG and mutants. R478A, D480A,F491A and T493A are the mutants for the potential iso-ADPr binding sites, and the rest are the mutants for the active site. (c) Quantified PARG activity by 1 min PARG TLC assay for wt mPARG and mutants. The activities are normalized to wt mPARG. Error bars represent standard deviation (n = 3). (d) The signature loops of the wt mPARG and E748 and E749 mutants. Wt mPARG in blue; E748N in orange; E749N in magenta; E748Q in green; E749Q in cyan. The side chains for residues 748 and 749 are shown in sticks.

Figure 3. A potential secondary iso-ADPr binding site.

(a) A possible secondary binding site. iso-ADPr is showed in cyan stick. The bound iso-ADPr is close to the Exon4+5 encoded region (highlighted in black). (b) The 2Fo-Fc simulated annealed omit map of the potential secondary iso-ADPr binding region, calculated using the CNS package and contoured at 1.5σ. The iso-ADPr was omitted and simulated annealing was performed to remove model bias prior to electron density calculation. It is also apparent that the iso-ADPr molecule in this position is not restricted by crystal packing.

Figure 4. N-terminal exon4+5 encoded regulartory segment.

(a) Exon 4+5 encoded segment docks on hydrophobic groove of the back side of mPARG catalytic domain. The core of mPARG is shown as grey surface. The exon 4 encoded region is shown in red, while the exon 5 encoded region is shown in blue. Met454, Met457, Leu464 and Leu467 are highlighted in orange sticks. (b) B factor spectrum of vertebrate PARG structures. The regions with low B factor are in cyan, and the ones with high B factor are in red. The exon4+5 encoded segments of mPARG, rPARG (PDB: 3UEK) and hPARG (PDB:4B1G) all have a relatively higher B factor than the core region of PARG catalytic domain. (c) In trans complementation PARG TLC assays with increasing peptide : PARG ratios.

Figure 5. A model for different modes/stages of PAR degradation by PARG.

Based on the data from this study and previous studies, we propose the catalytic mechanism of PAR degradation by PARG. In the early stage, PARG randomly recognizes the PAR between (n-1) and (n) ADPr, then hydrolyzes the glycosidic bond in-between (endo- activity). Because the (n-1) PAR polymer has lower binding affinity, it leaves PARG after the reaction. Long PAR polymers may have higher affinity with PARG than short PAR, due to the interaction between the (n+x) ADPr unit and the potential secondary iso-ADPr binding site. The (n+) PAR polymer stays with PARG after cleavage. Thereafter, PARG can slide along the (n+) PAR polymer to cleave ADPr units from proximal to distal end one by one (exo- activity). In the late stage, when the PAR polymer is not long enough, which result in the lower binding affinity with PARG (no secondary binding site), PARG can no longer processively degrade PAR polymers. Shorter PAR leaves PARG after every single cleavage, and is degraded by PARG distributively.