In the present study, we describe the ligand-bound structures of the ARH family members ARH1 and ARH3. Our data indicate that despite the highly specialized reactions they catalyze, with ARH1 hydrolyzing ADP-ribosyl linkages to arginine and ARH3 to serine residues (), these enzymes are very similar, in particular with respect to their active sites. The structures presented here provide critical insights into their mode of enzyme-ligand interaction, help us to understand the differences in their catalytic behavior, and present a useful tool for targeted drug design.

One major distinction between the structures of ARH1 and ARH3 is the coordination of the adenosine pyrophosphate moieties, which has direct consequences for their substrate interactions. The ability of ARH3, but not ARH1, to degrade PAR chains () has implicated it in PARP1-mediated cell death, where it guards against cell death through its ability to degrade PAR chains (). Our structures suggest that ARH3 is able to bind and cleave PAR chains both in an endo and exo manner, thus allowing the degradation of both attached and free chains. This is due to the orientation of the proximal ribose, which exposes both the 2′ and 3′ OH toward the enzyme surface, with hardly any limitations to the attachment of further ADPr units. In contrast, the proximal ribose in ARH1 is coordinated by the rigid adenosine binding loop (loop 16). The resulting orientation aids selectivity toward MARylated substrates, which aligns well with previous reports that ARTCs are mono-specific transferases ().

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Figure 4 Proposed Reaction Mechanism for ARH3

Given the different substrate specificities, ARH1 preferentially cleaves N-glycosidic and ARH3 O-glycosidic bonds, the similarities between the active centers is surprising. All major contacts needed for the orientation of the distal ribose appear to be conserved, which includes the coordinated Mgions as well as interaction with Ala98 and Asp302 (hARH1) and Gly101 and Asp303 (LchARH3), respectively. However, the 1″ OH in the LchARH3:ADPr complex appears to take part in the second coordination sphere of Mg, whereas this is not the case in the hARH1:ADPr complex. In the latter, the ADPr takes on a more relaxed conformation, which is mimicked in the LchARH3:Arg-ADPr and :ADP-HPD complexes, thus suggesting that an α-linked oxygen draws the ribose ring closer to the magnesium ion. A second striking aspect of the Mgcoordination is the placement of an absolutely conserved glutamic acid crucial for full catalytic activity (Glu25 in hARH1 and Glu33 in LchARH3, Figures S3 and 2 C). While this glutamate coordinates Mgin the earlier reported human apo structure (PDB: 2FOZ ) (), our data show that it is not strictly necessary for Mgcoordination ( Figures 1 E and 2 A). During revision, structures of the hARH3:ADPr complex became available, highlighting an even greater degree of flexibility in glutamic acid positioning ( Figure S1 C) (). While the mode of ADPr coordination between hARH3 and LchARH3 is very similar overall, a key differences is the coordination of 2″OH at the distal ribose. In our structures, this moiety is either coordinating Mg(ADPr structure) or bridging between the Mgions (ADP-HPD, Arg-ADPr, and IDPr). In contrast, the distal ribose of the hARH3:ADPr complex is rotated away from the binuclear metal center allowing the bridging water of the apo form to remain albeit in very close proximity (2.14 ± 0.11 Å) to the 2″ OH group. It is interesting to note that in hARH1 Glu25 is part of the short helix α2, which is replaced by a short loop with Glu33 positioned at the end of helix α1 in LchARH3. This exchange imposes different constraints on the flexibilities of these residues during catalysis. Finally, the structure of the LchARH3:ADPr complex from the initial crystal system prior to optimization (PDB: 6G1Q , this study) suggests that while ligand binding is possible in the absence of Mg, the specific arrangement of ADPr cannot be maintained in the active site. The low Kof magnesium on its own (∼5.4 mM for hARH3, ∼2.3 mM for LchARH3; Figures 2 F and 2G), together with the apparent low concentration needed to restore activity (∼50 μM, Figure S2 C), suggests that the substrate contributes to the stabilization of the binuclear metal center. This dissociative tendency is highlighted by the structures of hARH1:ADP-HPM and LchARH3:ADP-HPM, which lack Mgin their active site, despite the presence of magnesium in the final crystallization condition (50 mM and 10 mM, respectively). The inability to coordinate Mgcould account for the dramatic drop in Kand ICobserved for ARH3 ( Table 3 ). This mode of inhibition stands in contrast to the structural studies on ADP-HPD as a PARG inhibitor (). In PARG, the substrate is bound in a strained conformation due to the presence of a conserved phenylalanine (Phe875 in human PARG), which forces the distal ribose, in particular the linkage 1″ carbon, toward the catalytic loop. This conformation is further stabilized by extensive contacts between the Phe875 containing loop and the pyrophosphate moiety. Together this leads to a closed conformation and restricts the possibility of movement in the distal ribose. Consequently, comparison of ADPr (PDB: 4B1H ), ADP-HPD (PDB: 4B1J ), and ADP-HPM (this study; Figure S2 D, Tables 1 and S1 ) -bound PARG structures revealed little difference in the mode of binding ( Figures S7 A and S7C). The strained conformation, however, brings the ribose ring oxygen into the proximity of the Pphosphate, which could stabilize the oxocarbenium reaction intermediate and explain the higher potency of ADP-HPD, which has a positively charged pyrrolidine nitrogen, in comparison with ADPr. In contrast, both ARH1 and ARH3 complexes show a relaxed ADPr conformation ( Figures 1 E, S2 A, and S2B), which precludes a theoretical oxocarbenium-phosphate interaction. Together with the few restrictions on movement of the distal ribose in the active site, these findings offer several potential explanations: (1) formation of a short-lived oxocarbenium intermediate, (2) movement of the substrate during the catalytic mechanism, or (3) a mechanism that does not involve the formation of an oxocarbenium intermediate. While we cannot rule out (1) or (2), our findings and earlier reported data support a mechanism in which the distal ribose is orientated in the active site via interactions with both Mgions ( Figure 4 ). In this strained conformation, the scissile bond becomes susceptible to a base-mediated S2 attack of a nucleophilic water from the second coordination sphere. The intermediate serine oxyanion is stabilized by the interaction with Mg, while the distal ribose obtains a more relaxed conformation and leaves the immediate catalytic site. The serine is subsequently protonated and released. Given the importance of Glu33 for the catalytic mechanism, its high flexibility ( Figure S1 C) and no alternative reversible protonatable residues in the catalytic center, we propose that it acts as acid/base in the reaction cycle and assists the serine release by coordinating Mgafter the protonation step.