A dynamic Asp–Arg interaction is essential for catalysisin microsomal prostaglandin E2 synthaseJoseph S. Brocka,1, Mats Hamberga,1, Navisraj Balagunaseelana, Michael Goodmanb, Ralf Morgensternc,Emilia Strandbacka, Bengt Samuelssona,2, Agnes Rinaldo-Matthisa, and Jesper Z. Haeggströma,2

aDepartment of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden; bDepartment of Chemistry, Vanderbilt UniversitySchool of Medicine, Nashville, TN 37232-6304; and cInstitute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden

Contributed by Bengt Samuelsson, December 18, 2015 (sent for review October 9, 2015; reviewed by Lawrence J. Marnett, Charles N. Serhan, andTakao Shimizu)

Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsiblefor the formation of the potent lipid mediator prostaglandin E2 underproinflammatory conditions, and this enzyme has received consider-able attention as a drug target. Recently, a high-resolution crystal struc-ture of human mPGES-1 was presented, with Ser-127 being proposedas the hydrogen-bond donor stabilizing thiolate anion formationwithin the cofactor, glutathione (GSH). We have combined site-directedmutagenesis and activity assays with a structural dynamics analysis toprobe the functional roles of such putative catalytic residues. We foundthat Ser-127 is not required for activity, whereas an interaction be-tween Arg-126 and Asp-49 is essential for catalysis. We postulate thatboth residues, in addition to a crystallographic water, serve critical roleswithin the enzymatic mechanism. After characterizing the size orcharge conservative mutations Arg-126–Gln, Asp-49–Asn, and Arg-126–Lys, we inferred that a crystallographic water acts as a generalbase during GSH thiolate formation, stabilized by interaction withArg-126, which is itself modulated by its respective interaction withAsp-49. We subsequently found hidden conformational ensembleswithin the crystal structure that correlate well with our biochemicaldata. The resulting contact signaling network connects Asp-49 to distalresidues involved in GSH binding and is ligand dependent. Our workhas broad implications for development of efficient mPGES-1 inhibitors,potential anti-inflammatory and anticancer agents.

inflammation | prostaglandin | mPGES-1 | MAPEG | mechanism

Prostaglandin E2 (PGE2) is an abundant lipid mediator thatsignals via four receptors (EP1–4) to induce an array of important

biological actions in physiology as well as pathophysiology (1). Underproinflammatory conditions, biosynthesis of PGE2 proceedsfrom arachidonic acid, which is converted to the unstable endoper-oxide PGH2 by cyclooxygenase type 2 (COX-2). PGH2 is furtherisomerized into PGE2 by microsomal PGE synthase type 1 (mPGES-1) (2, 3). mPGES-1 is encoded by PTGES and is up-regulated bymitogens and cytokines in a pathway that is functionally coupled toCOX-2 (2, 4). Because of its key role in PGE2 synthesis, mPGES-1has attracted attention as a potential drug target in the areas ofinflammation, pain, fever, and cancer (5).mPGES-1 is a member of the MAPEG (Membrane-Associated

Proteins in Eicosanoid and Glutathione metabolism) superfamily ofenzymes (6), which also includes two key proteins in the leukotriene(LT) cascade, viz. 5-lipoxygenase activating protein and LT C4 syn-thase (LTC4S). All MAPEG members are integral, homotrimericmembrane proteins, and structural information on this family hasbeen scarce. However, significant progress has recently been made inthis area with several high-resolution structures being solved by X-raycrystallography (7–9). In particular, the crystal structures of humanLTC4S provided detailed structural information, including an argi-nine residue that was later shown to activate the glutathione (GSH)thiolate (10, 11). We have previously proposed that this conservedarginine residue is also essential for enzymatic activity in mPGES-1 asArg-126 (12). The recent structural determination of mPGES-1,however, at an exceptionally high resolution of 1.16 Å, uncoveredseveral unanticipated structural features (13). The active sites, found

at the three monomeric interfaces, show that Ser-127 is positionednear the GSH thiol group, indicating that it may act as a hydrogen-bond donor to assist in thiolate formation during catalysis. Further-more, Arg-126 and Asp-49 participate in a charge interaction thatcould also contribute to catalysis. This structural information issupported by a mesophase crystal structure of an engineered versionof mPGES-1 (14) and several inhibitor complexes that have recentlybeen published (15).Here, we initially confirmed the necessity for GSH thiolate during

catalysis via incubations with the analogous tripeptide γ-Glu–Ser–Gly(GOH). We then used site-directed mutagenesis to analyze thefunctional roles of active site residues. We found that Ser-127 isnonessential for catalysis, whereas Arg-126 and Asp-49 are crucialand mutually dependent for native isomerase activity of the enzyme.Because the latter codependence of activity could be rationalized bya dynamic functional role of these residues, we turned to the high-resolution X-ray data (13, 15) deposited in the Protein Data Bank(PDB; www.rcsb.org) (16) to provide evidence of their dynamicmotion within the crystal structure. Several recent studies haveshown that the information present in such data is often under-estimated and that it is possible to refine multiple conformations ofresidues simultaneously, each with individually refined occupancyand B factors, without overfitting the data (17–22). By such samplingof low-level electron density, discrete, “hidden” conformations arerevealed, facilitating a more quantitative representation of dynamic

Significance

Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is anintegral membrane protein that produces prostaglandin E2 (PGE2),a mediator of inflammation, fever, pain, and tumorigenesis. Herewe show that a serine residue implicated by the crystal structureis not required for function, whereas an arginine and aspartateresidue in the active site, observed to be interacting within thecrystal structure, are essential and mutually dependent duringcatalysis. We also demonstrate that a contact signaling networkcan interrupt the arginine–asparagine interaction and facilitatetheir participation in the chemical mechanism. Our work hasbroad implications for development of effective mPGES-1 inhib-itors, potential drugs with clinical application in treatment of in-flammatory diseases and cancer.

Author contributions: M.H., B.S., A.R.-M., and J.Z.H. designed research; J.S.B., M.H., N.B.,and E.S. performed research; M.G. and R.M. contributed new reagents/analytic tools;J.S.B., M.H., N.B., A.R.-M., and J.Z.H. analyzed data; and J.S.B. and J.Z.H. wrote the paper.

Reviewers: L.J.M., Vanderbilt University Medical Center; C.N.S., Brigham and Women’sHospital/Harvard Medical School; and T.S., National Center for Global Healthand Medicine.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1J.S.B. and M.H. contributed equally to this work.2To whom correspondence may be addressed. Email: jesper.haeggstrom@ki.se or bengt.samuelsson@ki.se.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1522891113/-/DCSupplemental.

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motion within the crystal lattice. Furthermore, it has been shown thatthis information is often essential for understanding enzymaticfunction (23) and mechanism (24, 25) and successfully achievingstructure-based drug design (26).We quantified the dynamic conformations of active site residues

using the software qFit (20) and CONTACT (27). This methodgenerated significant improvements in the quality indicators forPDB ID codes 4AL0 and 4AL1 [1.16 and 1.95 Å, respectively (13)]and revealed a ligand-dependent contact network that corroboratesthe mechanism suggested from biochemical data. These van derWaals interactions within the binary complex with GSH (PDB IDcode 4AL0) reveal an extensive network of correlated side-chainmotions within the cytoplasmic “C-domain” that forms the bottomof the active site and confirm a dynamic role of Asp-49 in catalysis.In comparison, the much smaller networks found in PDB ID code4AL1, with a bisphenyl–GSH analog and detergent moleculebound in the active site, indicate that ligand binding can influence

network signaling. This finding is also supported by our analysis ofrecently published inhibitor complexes (15).Our results suggest that the positively charged Arg-126 stabilizes

transient thiolate formation and that its dynamic interaction withAsp-49 is essential for catalysis. We also observed a crystallo-graphic water molecule that is ideally situated to act as a protonacceptor during this process. Furthermore, we found a strikingcontact signaling network within the active site that effects theconformation of residues in a ligand-dependent fashion.

Results and DiscussionGSH Thiolate Is Essential for Catalysis. Fig. 1 depicts a substrate-limited assay that measured absolute product formation (nano-grams) as described in Materials and Methods. Incubations withnative enzyme in the presence of GSH resulted in near total con-version of PGH2 to PGE2 and are concurrent with the specificactivity previously reported in the literature (∼4 μmol·min−1·mg−1 at0 °C) (28). A negative control involved microsomal preparationsof WT mPGES-1 resuspended in buffer containing the GSHanalog, GOH, that differs from the native cofactor only by thereplacement of the thiol moiety by a hydroxyl group and did notproduce product above background levels. This finding providesstrong evidence that thiolate anion is the chemical species of thecofactor essential for catalysis.

Ser-127 Is Nonessential for Catalysis. Judging from the orientationof Ser-127 in the recently published crystal structure (13), theauthors’ hypothesis that its hydroxyl group may act as a hydrogen-bond donor to stabilize a GSH thiolate is apt, because this mech-anism of thiol activation is a common theme within the evolution ofsoluble GSH transferases (29). However, because we had pre-viously proposed Arg-126 as a strong candidate for this role (12),we investigated the function of Ser-127 in the conversion of PGH2into PGE2.To detail the role of Ser-127 in mPGES-1, we exchanged this

residue for an alanine by site-directed mutagenesis. After expres-sion in Pichia pastoris and purification, aliquots of recombinantprotein were incubated with PGH2. Formation of PGE2 was ana-lyzed by GC-MS. The combined measurements obtained from atleast three different preparations of enzyme are depicted in Fig. 1and show that Ser-127–Ala exhibits the same level of PGE2 syn-thase activity as WT mPGES-1. This finding was true for bothpurified and microsomal preparations of the enzyme (Fig. 1). Inaddition, the dual conformations observed for this residue in the

Fig. 1. Mutagenic analysis of mPGES-1 active site residues. Aliquots of WT,S127A, D49N, R126Q, and R126KmPGES-1 were incubated with 12 μMPGH2 andanalyzed for PGE2 formation by GC-MS, as described inMaterials and Methods.The total amount (nanograms) of PGE2 formed is shown from both purified andmicrosomal preparations of enzyme. PGE2 formation was also monitored formicrosomal preparations of WT enzyme incubated with GOH. Values representthe combined measurements from at least three different preparations of en-zyme (n = 3), with error bars representing their SD. The levels of PGF2α formedwere also measured by this method as shown in Fig. S1.

Fig. 2. The active site architecture of mPGES-1. The active sites of PDB ID codes 4AL0 (A) and 4AL1 (B) are compared with post-qFit conformational fitting and re-finement. The coordinates of PDB depositions are overlaid in translucent over the refined qFit ensembles shown as opaque conformers in stick representation. The 2mFo-DFc electron density corresponding to a mechanistically relevant solvent molecule is shown as blue mesh contoured at 1 rmsd, and the rotation plane of the R126guanidinium is shown relative to the carboxylate of D49 (44.7°) (A). This water is absent within the qFit-refined bis-phenyl complex (B), potentially due to a reducedcapacity for GSH thiol interaction. Amolecule of octyl glucoside bound at the C-domain and low occupancy GSH (∼13%)within the active site of B have been omitted forclarity (cf. Fig. S3). Polar interactions are shown as dashed lines with distances given in Å.

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crystal structure indicate the absence of a strong hydrogen-bondinginteraction. Conversely, Arg-126 is observed in a single confor-mation with an Nη-GSH thiol distance of 3.4 Å. We believe thatthis active site geometry also substantiates strong evidence for amechanism of GSH thiol activation by an Arg-126 guanidiniuminteraction (30). Hence, despite compelling structural evidence,Ser-127 does not play a critical role in mPGES-1 catalysis.

Mutation of Arg-126 and Asp-49 Compromises PGE2 Synthase Activity,but Allows PGH2 Reduction to PGF2α. In light of the new structuraldata (13), we wanted to reexamine the functional role of Arg-126and mutated this residue into both a glutamine and a lysine residueusing site-directed mutagenesis. According to the crystal structureof mPGES-1 (13), Arg-126 and Asp-49 participate in an inter-monomeric charge interaction. Therefore, we also mutated thenegatively charged counterpart, Asp-49, into an asparagine residue.We anticipated that the size and charge conservative mutations ofthese residues could serve in probing their role in the enzymatic

mechanism, while minimizing steric and electrostatic repulsioneffects, such as disruption of the monomer interface. Although wealso attempted to create the charge conservative mutant Asp-49–Glu, the resulting transformed construct failed to express, pre-sumably because it resulted in an unstable quaternary structure.After solubilization with detergent and purification via Ni-affinity

chromatography, these mutants were assayed for PGE2 synthaseactivity as described above. For three different purifications of eachisoform, we found that the mutated enzymes did not convert PGH2into PGE2 above background levels. After preparations of micro-somal fractions, however, we found that the charge conservativemutation Arg-126–Lys still retained a low level of isomerase activity,indicating that a native membrane environment and a formal posi-tive charge at position 126 are important factors for catalysis (Fig. 1).From these results, we conclude that both Arg-126 and Asp-49 arekey to the PGE2 synthase activity of mPGES-1.That both of these residues are essential for catalysis is intriguing,

because one could expect Arg-126 to be precluded from participating

Fig. 3. Contact signaling within mPGES-1. (A) The van der Waals contact network identified by qFit conformational fitting and subsequent CONTACT analysisof mPGES-1 PDB entries are shown with translucent molecular surface representations over alternate conformers and correspondingly colored node diagrams.The nodes are connected by edges whose width is weighted according to the number of networks involving the pair they connect. The network observedwithin PDB ID code 4AL0, which includes the active site residue D49, is shown in red, both from the perspective of the membrane plane (Left) and the cy-toplasm (Right). (B) The corresponding contact networks identified from the qFit ensemble complex within the bis-phenyl GSH complex (PDB ID code 4AL1)are much smaller and are shown in cyan and red. The latter contains the active site residue R126, now observed in dual conformations, possibly due tonegation of GSH thiol interaction. One possible pathway is show in more detail within Movie S1.

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in thiolate stabilization if it was already engaged in a stable salt bridgeinteraction with Asp-49. Analysis of the relative torsional angles,however, shows the out-of-plane angle of the Asp-49 carboxylaterelative to the Arg-126 guanidinium to be 44.7° (Fig. 2A). This valueis far in excess of the ∼8–10° found to be typical of bidentate inter-actions for structures of a similar resolution as reported in a recentcomprehensive review (31). Therefore, the Asp-49–Arg-126 in-teraction cannot be classified as the energetically stable, bidentateinteraction of a formal salt bridge and implies that the energeticbarrier for its disruption would be low. This finding provides evidencefor the capacity of these residues to dynamically participate in activesite chemistry and is corroborated by conformational fitting withqFit (27), which reveals hidden conformations of both residuesdepending on the identity of the adjacent ligand (Fig. 2).As we had previously observed for Arg-126 mutants (12), we

found that other catalytically inactive mutants assayed in thisstudy displayed a promiscuous reductase activity, convertingPGH2 into PGF2α. Notably, the most pronounced activity in thisrespect was again observed for microsomal preparations of theArg-126–Lys mutant (Fig. S1).We confirmed that all mutants possessed the same tertiary fold

as native enzyme via comparison of circular-dichroism spectra(Fig. S2).

A Crystallographic Water Molecule Is Ideally Situated to Participate inthe Mechanism. Analysis of the active site architecture also sug-gests that the α-carboxylate of GSH is involved in thiolate for-mation, via a tightly bound crystallographic water (2Fo − Fc peakof ∼5 rmsd, ADP = 21.9 Å2) within the active site (Fig. 2A). Byforming a hydrogen-bonding network from the α-carboxylatemoiety of GSH to its thiol group, it is ideally placed to assist indeprotonation of the latter during catalysis. The pKa of theα-carboxylate, in turn, is undoubtedly lowered by the side-on,out-of-plane interaction with the guanidinium of Arg-38 (torsionangle 45.9°), which is itself engaged in solvent-mediated inter-actions with the main-chain carboxyl groups of Ala-43 and Arg-60. This architecture is highly reminiscent of the “electron-sharingnetwork” that is functionally conserved in all classes of solubleGSTs for the same purpose (32), and the use of a bridging watermolecule to transfer the thiol proton to the α-carboxylate ofGSH has been shown to be energetically favorable within analpha class soluble GST (33). Crucially, density for this water isabsent for the Phenix (34) refined bis-phenyl GSH complex(PDB ID code 4AL1), in which the relative occupancies to GSHwere refined as 0.87:0.13, respectively. After conformational changeof Asp-49, we hypothesize that Arg-126 can further decrease the

GSH thiol pKa via charge stabilization. The crystallographicwater molecule could then function as the yet-unidentified basethat accepts a proton from GSH during thiolate formation,concurrently forming a transient hydronium ion or shuttling theproton to the α-carboxylate. Once formed and stabilized by in-teraction with Arg-126, we expect attack of GSH thiolate uponthe endoperoxide ring at the C-9 position, resulting in O–O bondcleavage and proton donation via the hydronium ion. Asp-49,liberated from its interaction with Arg-126, would now be free tofunction as a base within the resulting transition state, facilitatinga decrease of the C-9 proton pKa, and spontaneous decompositionto yield the product PGE2 and regenerated GSH (Fig. 4). Althoughan alternative mechanism in which thiolate would act as a generalbase abstracting the C-9 proton has been suggested to be moreenergetically favorable in model systems (35, 36), the probabilityof either pathway would ultimately be determined by the preciseorientation of substrate relative to cofactor within the enzymaticactive site. Although the apparent ability of the active site mu-tants characterized here to produce PGF2α via reduction of aputative sulphenic acid ester intermediate speaks in favor of theformer (Fig. S1), this alternative mechanism is shown in Fig. S4.

A Contact Signaling Network Modulates Active Site Residues in mPGES-1.We submitted the PDB entries associated with the recently publishedcrystal structure of mPGES-1 (PDB ID codes 4AL0 and 4AL1)(13) to the qFit server (smb.slac.stanford.edu/qFitServer/) (20). Thissoftware automatically samples conformational heterogeneity that isinterpretable by fitting partial occupancy conformational ensemblesinto low-level electron density. The CONTACT algorithm was thenused to calculate resulting van der Waals contact networks that in-dicate a probable correlation of conformations at each site (27).Post-qFit conformational fitting and subsequent refinement by

Phenix (34) of the 1.16-Å binary complex with GSH (PDB ID code4AL0) resulted in a small, but significant, improvement of structurequality indicators, including the decrease of R/Rfree values from12.2/13.0% to 11.6/12.8%, respectively. Subsequent analysis of thestructure with CONTACT revealed an extensive network of cor-related side chain interactions centered upon the short, cytoplasmichelix separating transmembrane helices I and II that was referred toby Sjögren et al. (13), and will be hereafter, as the C-domain. Ofmost interest is that the network facilitates signal transduction fromresidues involved in the recognition of GSH to the active site res-idue Asp-49. This process could facilitate the disruption of itsinteraction with Arg-126, facilitating the latter’s role in thiolatestabilization on a time scale specific to catalysis. Thr-34 and Leu-69,located on helices I and II, respectively, make hydrophobic contactswith the γ-glutamyl moiety of GSH and initiate series of correlatedvan der Waals overlaps that ultimately affect Asp-49, e.g., Leu-69→Thr-34 → Cys-68 → Asp-64 → Lys-41 → Arg-40 → Leu-39 → His-53 → K42 → H53 → Asp-49 (Fig. 3A and Movie S1).Conversely, Arg-126, with which it forms an intermonomeric

interaction, is fitted as the single conformation observed within thecrystal structure (13) (Fig. 2A). As discussed above, we believe thisactive site geometry is strong evidence of a GSH thiol–Arg-126interaction. Although the pKa of GSH thiol has been measured as9.42 in solution (37), the dynamic interaction of GSH thiol withArg-126, combined with the solvent restricted electrostatics of theactive site, may allow GSH to transiently form thiolate during ca-talysis via a mechanism of charge redistribution. Specifically, thehydrogen-bonding network formed by a crystallographic watermolecule between the α-carboxylate and thiol moieties of GSHmay be crucial in this respect (Figs. 2A and 4).This finding is corroborated by comparison with the qFit-

generated structural ensembles of the bis-phenyl complex (PDBID code 4AL1), in which Arg-126, now with a reduced potentialfor interaction with thiol, is observed to be in dynamic motion(Figs. 2B and 3B) (see below).

Fig. 4. Proposed mechanism of mPGES-1. For details, please see Results andDiscussion.

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Contact Signaling Is Ligand-Dependent. After an iterative fitting ofalternate conformations with qFit (20), building of N-terminalresidues into density, and subsequent refinement with Phenix(34) (described in Materials and Methods), a significant im-provement of quality indicators was achieved for the 1.95-Åresolution mPGES-1 complex with bisphenyl–GSH (PDB IDcode 4AL1), with a reduction of R/Rfree values from 16.3/17.2%to 13.7/16.6%, respectively. Subsequent CONTACT analysislacked the extensive signaling network found within the GSHcomplex, however, which were instead focused on opposing sidesof the active site. The ensemble structure was found to containtwo networks of four and five residues, respectively, the latter ofwhich occurs in the cytoplasmic loop between helices III and IVand contains alternate conformations of Arg-126 (Fig. 3B). Thisfinding suggests that the activation of dynamic contact networksin mPGES-1 may be dependent upon the identity of the ligandbound at the active site. Although the difference in structuralinformation inherent in the two datasets (1.16 Å cf. 1.95 Å)should be considered when drawing comparisons between theqFit-generated ensemble structures, the resolution of the 4AL1dataset is still significantly higher than the upper limit of 2.1 Åsuggested by the software developers (smb.slac.stanford.edu/qFitServer/) (20). In addition, we performed a qFit/CONTACTanalysis of high-resolution (1.41–1.52 Å) mPGES-1 inhibitorcomplexes recently published (15) (PDB ID codes 4YK5, 4YL0,4YL1, and 4YL3). These four compounds are also observed tobind in the intermonomeric active site, making extensive contactwith the C-domain. Although the four inhibitors are varied instructure and binding modes, they all share a common interactionwith the C-domain and lack the extensive networks found in theholoenzyme complex with GSH (PDB ID code 4AL0). Intriguingly,the same interaction is also fulfilled by an octyl glucoside (n-octyl-β-D-glucoside) detergent molecule (not shown in Figs. 2 and 3 for clarity;cf. Fig. S3) within the bis-phenyl complex (PDB ID code 4AL1),whose polar head group also makes contact with the turn/helixC-domain motif and whose hydrophobic tail stacks against the bis-phenyl moiety of the GSH analog (13) (Fig. S3). This finding indi-cates that stabilizing contacts within this region may disrupt potentialfor signal transduction (Fig. S3). As shown in Fig. 3A and Movie S1,the dynamic conformations of Lys-41, Arg-40, Leu-39, and His-53are essential to the transmission of the contact network within theC-domain, and ultimately to the active site residue, Asp-49. There-fore, it is possible that their mode of inhibition is mediated by fa-voring certain conformations of these residues from the structuralensemble and subsequent interruption of signaling (26).This mechanism could be a common theme of potent mPGES-1

inhibitors. In a recent analysis of binding sites via mass spectrometryhydrogen/deuterium exchange experiments (38), the authors foundthat the greatest differences common to the twomost potent inhibitorswere observed in residues 37–54, corresponding to the C-domain.

ConclusionsThe combined results of site-directed mutagenesis, functionalassays, structural ensemble, and contact network analysis pre-sented herein provide strong evidence for a mechanism of PGE2synthesis by mPGES-1 that features an activation of GSH thio-late by Arg-126, modulated via its respective interaction withAsp-49. Furthermore, we show that conformations of the lattercan be affected by a ligand-dependent contact signaling, con-necting it to distal residues involved in GSH recognition, with the

potential to dynamically alter the Asp-49–Arg-126 interactionduring catalysis (Fig. 3).We propose a previously unidentified mechanism of PGH2 isom-

erization by mPGES-1 that features a prominent role of a water-mediated interaction with the α-carboxylate of GSH and an Asp-49–mediated thiolate stabilization by Arg-126 (Fig. 4). We hypothesizethat the active site of mPGES-1 lowers the pKa of GSH thioland the C-9 proton of PGH2 concurrently via respective interac-tions with Arg-126 and Asp-49, facilitated by their dynamic con-formational change in response to contact network signaling.Charge conservation in this solvent-restricted environment couldthus be achieved via proton shuffling by the crystallographicwater/α-carboxylate hydrogen-bonding network (Fig. 4).This work has broad implications for the pharmacological efforts

to inhibit this enzyme, which are a current topic of discussionwithin the literature (39).

Materials and MethodsProtein Expression and Purification. Recombinant wild-type (WT) and active-sitemutants of human mPGES-1 were overexpressed in P. pastoris and purified byNi-affinity chromatography before exchanging buffer to 0.1 M phosphatebuffer, 0.03% dodecyl maltoside, and 2.5 mM GSH, pH 7.4. Microsomal prep-arations were prepared via ultracentrifugation of lysed cell supernatant andhomogenization of the microsomal pellets in assay buffer (20 mM Tris·HCl, pH7.8, 2.5 mM GSH). For further details, please refer to SI Materials and Methods.

Synthesis of GOH. The oxygen analog of GSH, GOH, was synthesized in a three-step procedure based on a published method (40). For further details, pleasesee SI Materials and Methods.

Enzyme Activity Assay. Conversion of PGH2 to PGE2 by WT or mutatedmPGES-1 were quantified by using GC-MS as described (12). For furtherdetails, please refer to SI Materials and Methods.

Analysis of Dynamic Contact Networks. The qFit Web server (smb.slac.stanford.edu/qFitServer/) and the CONTACT algorithm (27) were used forthe quantification of conformational ensembles and functional contactnetworks, respectively. Before analysis, the physiological trimer was gener-ated from the asymmetric unit via crystallographic symmetry using theprogram COOT (41). For PDB ID codes 4AL0, 4YL0, 4YL1, 4YL3, and 4YK5, thecoordinates were submitted to the qFit server and refined and prepared forCONTACT as described (27), by using Phenix-1.9-1692 (34) without manualintervention. For PDB ID code 4AL1, significant density improvement at theamino terminus allowed residues 4–9 to be built into density after qFitconformer fitting and refinement. After a second round of refinement, theresulting improvement in quality indicators such as the Rfree value weresignificant, such that the improved phase estimates were anticipated toaffect the conformational ensemble fitting. Hence, the improved coordi-nates were resubmitted to the qFit server before being refined and pre-pared for analysis with CONTACT as above. Settings for all CONTACTanalyses were as follows: Tstress (percentile) = 0.4, max_path_length = 100,sc_only_flag = f (all atom), relief_threshold = 0.90.

ACKNOWLEDGMENTS. We thank Gunvor Hamberg for technical assistanceand gratefully acknowledge the late Richard Armstrong, who provided theGOH GSH analogue. Part of this work was performed at the KarolinskaInstitutet Protein Science Facility. Some computations were performed onresources provided by the Swedish National Infrastructure for Computingat Linköping University. This work was supported by Swedish Research Coun-cil Grant 10350 and CERIC Linnaeus Grant; the Stockholm County Council(Cardiovascular Program, Thematic Center Inflammation); and NovoNordiskFoundation Grant NNF15CC0018346. J.Z.H. is the recipient of a DistinguishedProfessor Award from Karolinska Institutet.

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