ADP-HPD inhibition and binding data for human PARG constructs.

<p>Replicates are indicated in parentheses. IC₅₀ values are arithmetic means ± standard errors. Values quoted for the SPR and ITC data are arithmetic means ± absolute errors calculated by propagation of errors. SPR binding constants were derived from steady state fits. n.d. = not determined.</p

hPARG catalytic domain constructs show equivalent <i>in vitro</i> enzymatic activity and ADP-HPD binding properties as the full-length enzyme.

<p>(a) Time-course of PAR-PARP1 hydrolysis by recombinant PARG as measured in a homogeneous time-resolved fluorescence (HTRF) assay. Data points are the mean of three measurements carried out on separate occasions. (b) Inhibition of PAR-PARP1 hydrolysis by ADP-HPD. Data points are the mean of all measurements from three separate experiments, each run in triplicate ± Standard Error. Percent inhibition was calculated with respect to “No Enzyme” and “No Inhibitor” controls. (c) Representative binding sensorgrams and steady state fits for ADP-HPD binding to immobilised hPARG, hPARG4 and hPARG26 monitored by SPR. (d) Representative binding isotherms showing binding of ADP-HPD and OA-ADP-HPD to hPARG4 monitored by ITC.</p

Crystallographic statistics for structures of orthorhombic hPARG catalytic domain (hPARG26).

<p>Crystallographic statistics for structures of orthorhombic hPARG catalytic domain (hPARG26).</p

Schematic structure-based mechanism for the reported endo- and exo-glycohydrolase activities in hPARG.

<p>Selected interacting residues and water molecules are shown, with H-bonds drawn as dashed lines. The terminal ADPR unit (R1 = H) or possibly also an internal ADPR unit (R1 = PAR) within a linear (R2 = linear chain) or branched (R2 = branch point) PAR chain bind to the hPARG catalytic site. Glu756 acts as the catalytic acid/base to effect cleavage of the scissile ribose” 1″-O-R2 bond, releasing shorter, linear PAR and possibly also de-branched PAR. The oxocarbenium ion intermediate undergoes nucleophilic attack by one of two water molecules via an inverting (Wat1, blue) or retaining (Wat2, red) mechanism, to generate ADPR (R1 = H, exo-glycohydrolysis), or possibly also shorter PAR (R1 = PAR, endo-glycohydrolysis).</p

hPARG construct design.

<p>The 29 PARG fragments synthesized and tested for soluble expression, PARG activity and crystallisation are shown in relation to full-length hPARG(1–976) (hPARG). A representative disorder prediction (RONN, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050889#pone.0050889-Yang1" target="_blank">[30]</a>) and a schematic of the secondary structure for hPARG26 are shown above the hPARG domain diagram (domain boundaries based on reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050889#pone.0050889-Botta1" target="_blank">[5]</a>). Sites determined as sensitive to trypsin in limited proteolysis experiments are indicated above the disorder prediction as *. Experimental outcomes are indicated to the right of each construct thus: × no soluble expression, √ low level soluble expression, √√ high level soluble expression, ∼ no crystals observed and ♦ crystals obtained. SER1 = surface entropy reduction patch 1 (K616A, Q617A, K618A). SER2 = surface entropy reduction patch 2 (E688A, K689A, K690A). MTS = putative mitochondrial targeting signal.</p

Crystallographic statistics for structures of monoclinic hPARG catalytic domain (SeMet-hPARG26).

a<p>Effective resolution limited to 2.3 Å by ice rings.</p>b<p>Model partially refined against peak data.</p

Binding of ADPR, ADP-HPD and OA-ADP-HPD is accompanied by conformational changes in the active site of human PARG.

<p>2Fo-Fc omit maps for bound ligand and waters are shown in blue contoured at 1σ. Pictures prepared using PyMol (Schrödinger, LLC). (a) hPARG26 in complex with ADPR. Electron density clearly reveals binding of the α-anomer. A tightly bound water molecule (Wat1, also present in the unliganded structure), positioned 3.1 Å from the ribose” anomeric carbon, has been proposed to act as a nucleophile during hydrolysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050889#pone.0050889-Slade1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050889#pone.0050889-Kim1" target="_blank">[25]</a>. The ribose” 1″-OH PAR attachment site lies 2.6 Å from Oε2 of the putative catalytic acid/base, Glu756 (see inset). Overlay of ADPR-bound (grey) and unliganded (pale-green) structures highlights closure of the conserved G⁸⁷³AFG loop over the di-phosphate moiety, and rotation of the Phe902 side-chain out of the adenine pocket upon ADPR binding. (b) hPARG26 in complex with the transition-state mimetic, ADP-HPD. As noted in the ADPR complex, a water molecule (Wat1) lies close to the anomeric carbon below the plane of the HPD-ring. In the ADP-HPD complex, a second water molecule (Wat2) lies 3.7 Å from the anomeric carbon above the plane of the HPD-ring and within H-bond distance (<2.3 Å) of the Glu756 side-chain (see inset). Both of these waters are also present in the unliganded hPARG26 structure. Either could generate product (ADPR) by nucleophilic attack on the transition-state after cleavage of the scissile bond. (c) hPARG26 in complex with OA-ADP-HPD. Overlay of OA-ADP-HPD-bound (grey) and ADP-HPD-bound (pale-green) structures highlights rotation of the Tyr-795 side-chain to accommodate the 8-<i>n</i>-octylamino moiety. (d) 2D structure depiction of compounds used in this study.</p

Mapping site-directed mutations onto the hPARG26 structure explains their effect on PARG activity.

<p>The hPARG26-ADP-HPD structure is shown in ribbon representation coloured to highlight important structural motifs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050889#pone-0050889-g003" target="_blank">Figure 3</a>. Bound ADP-HPD is drawn as sticks with carbon atoms in yellow. Side-chains of Trp814 and Pro472 are drawn as sticks, with the H-bond between the Trp814 indole NH and Pro472 backbone CO shown as a dashed black line. Mutated residues are drawn as spheres and coloured according to their effect on PARG activity from red (activity abolished) through white (no effect) to blue (activity enhanced). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050889#pone.0050889.s004" target="_blank">Table S1</a> for further details of individual mutations.</p