1. A study investigated residues proposed to be important for catalysis in microsomal prostaglandin E2 synthase (mPGES-1), which produces prostaglandin E2. 2. Mutation analysis found that while Ser-127 was not essential, Arg-126 and Asp-49 were crucial for mPGES-1's native activity of converting PGH2 to PGE2. Mutations to these residues greatly reduced or eliminated this activity. 3. Analysis of crystal structures revealed multiple conformations of residues and a contact signaling network between Asp-49 and other residues involved in substrate binding. This network was dependent on the bound ligand and may be important for the enzyme's catalytic

1. A dynamic Asp–Arg interaction is essential for catalysis
in microsomal prostaglandin E2 synthase
Joseph 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
a
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden; b
Department of Chemistry, Vanderbilt University
School of Medicine, Nashville, TN 37232-6304; and c
Institute 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, and
Takao Shimizu)
Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible
for the formation of the potent lipid mediator prostaglandin E2 under
proinflammatory 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 proposed
as the hydrogen-bond donor stabilizing thiolate anion formation
within the cofactor, glutathione (GSH). We have combined site-directed
mutagenesis and activity assays with a structural dynamics analysis to
probe the functional roles of such putative catalytic residues. We found
that Ser-127 is not required for activity, whereas an interaction be-
tween Arg-126 and Asp-49 is essential for catalysis. We postulate that
both residues, in addition to a crystallographic water, serve critical roles
within the enzymatic mechanism. After characterizing the size or
charge conservative mutations Arg-126–Gln, Asp-49–Asn, and Arg-
126–Lys, we inferred that a crystallographic water acts as a general
base during GSH thiolate formation, stabilized by interaction with
Arg-126, which is itself modulated by its respective interaction with
Asp-49. We subsequently found hidden conformational ensembles
within the crystal structure that correlate well with our biochemical
data. The resulting contact signaling network connects Asp-49 to distal
residues involved in GSH binding and is ligand dependent. Our work
has 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 that
signals via four receptors (EP1–4) to induce an array of important
biological actions in physiology as well as pathophysiology (1). Under
proinflammatory conditions, biosynthesis of PGE2 proceeds
from arachidonic acid, which is converted to the unstable endoper-
oxide PGH2 by cyclooxygenase type 2 (COX-2). PGH2 is further
isomerized into PGE2 by microsomal PGE synthase type 1 (mPGES-
1) (2, 3). mPGES-1 is encoded by PTGES and is up-regulated by
mitogens and cytokines in a pathway that is functionally coupled to
COX-2 (2, 4). Because of its key role in PGE2 synthesis, mPGES-1
has attracted attention as a potential drug target in the areas of
inflammation, pain, fever, and cancer (5).
mPGES-1 is a member of the MAPEG (Membrane-Associated
Proteins in Eicosanoid and Glutathione metabolism) superfamily of
enzymes (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, homotrimeric
membrane proteins, and structural information on this family has
been scarce. However, significant progress has recently been made in
this area with several high-resolution structures being solved by X-ray
crystallography (7–9). In particular, the crystal structures of human
LTC4S 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 conserved
arginine residue is also essential for enzymatic activity in mPGES-1 as
Arg-126 (12). The recent structural determination of mPGES-1,
however, at an exceptionally high resolution of 1.16 Å, uncovered
several unanticipated structural features (13). The active sites, found
at the three monomeric interfaces, show that Ser-127 is positioned
near 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 that
could also contribute to catalysis. This structural information is
supported by a mesophase crystal structure of an engineered version
of mPGES-1 (14) and several inhibitor complexes that have recently
been 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 the
functional roles of active site residues. We found that Ser-127 is
nonessential for catalysis, whereas Arg-126 and Asp-49 are crucial
and mutually dependent for native isomerase activity of the enzyme.
Because the latter codependence of activity could be rationalized by
a 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 dynamic
motion within the crystal structure. Several recent studies have
shown that the information present in such data is often under-
estimated and that it is possible to refine multiple conformations of
residues simultaneously, each with individually refined occupancy
and B factors, without overfitting the data (17–22). By such sampling
of low-level electron density, discrete, “hidden” conformations are
revealed, facilitating a more quantitative representation of dynamic
Significance
Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is an
integral membrane protein that produces prostaglandin E2 (PGE2),
a mediator of inflammation, fever, pain, and tumorigenesis. Here
we show that a serine residue implicated by the crystal structure
is not required for function, whereas an arginine and aspartate
residue in the active site, observed to be interacting within the
crystal structure, are essential and mutually dependent during
catalysis. We also demonstrate that a contact signaling network
can interrupt the arginine–asparagine interaction and facilitate
their participation in the chemical mechanism. Our work has
broad 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’s
Hospital/Harvard Medical School; and T.S., National Center for Global Health
and Medicine.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
J.S.B. and M.H. contributed equally to this work.
2
To 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.
972–977 | PNAS | January 26, 2016 | vol. 113 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1522891113

2. motion within the crystal lattice. Furthermore, it has been shown that
this information is often essential for understanding enzymatic
function (23) and mechanism (24, 25) and successfully achieving
structure-based drug design (26).
We quantified the dynamic conformations of active site residues
using the software qFit (20) and CONTACT (27). This method
generated significant improvements in the quality indicators for
PDB ID codes 4AL0 and 4AL1 [1.16 and 1.95 Å, respectively (13)]
and revealed a ligand-dependent contact network that corroborates
the mechanism suggested from biochemical data. These van der
Waals interactions within the binary complex with GSH (PDB ID
code 4AL0) reveal an extensive network of correlated side-chain
motions within the cytoplasmic “C-domain” that forms the bottom
of the active site and confirm a dynamic role of Asp-49 in catalysis.
In comparison, the much smaller networks found in PDB ID code
4AL1, with a bisphenyl–GSH analog and detergent molecule
bound in the active site, indicate that ligand binding can influence
network signaling. This finding is also supported by our analysis of
recently published inhibitor complexes (15).
Our results suggest that the positively charged Arg-126 stabilizes
transient thiolate formation and that its dynamic interaction with
Asp-49 is essential for catalysis. We also observed a crystallo-
graphic water molecule that is ideally situated to act as a proton
acceptor during this process. Furthermore, we found a striking
contact signaling network within the active site that effects the
conformation of residues in a ligand-dependent fashion.
Results and Discussion
GSH 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 with
native enzyme in the presence of GSH resulted in near total con-
version of PGH2 to PGE2 and are concurrent with the specific
activity previously reported in the literature (∼4 μmol·min−1
·mg−1
at
0 °C) (28). A negative control involved microsomal preparations
of WT mPGES-1 resuspended in buffer containing the GSH
analog, GOH, that differs from the native cofactor only by the
replacement of the thiol moiety by a hydroxyl group and did not
produce product above background levels. This finding provides
strong evidence that thiolate anion is the chemical species of the
cofactor essential for catalysis.
Ser-127 Is Nonessential for Catalysis. Judging from the orientation
of Ser-127 in the recently published crystal structure (13), the
authors’ 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 of
soluble 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 PGH2
into 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 recombinant
protein were incubated with PGH2. Formation of PGE2 was ana-
lyzed by GC-MS. The combined measurements obtained from at
least three different preparations of enzyme are depicted in Fig. 1
and show that Ser-127–Ala exhibits the same level of PGE2 syn-
thase activity as WT mPGES-1. This finding was true for both
purified and microsomal preparations of the enzyme (Fig. 1). In
addition, 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 R126K mPGES-1 were incubated with 12 μM PGH2 and
analyzed for PGE2 formation by GC-MS, as described in Materials and Methods.
The total amount (nanograms) of PGE2 formed is shown from both purified and
microsomal preparations of enzyme. PGE2 formation was also monitored for
microsomal preparations of WT enzyme incubated with GOH. Values represent
the combined measurements from at least three different preparations of en-
zyme (n = 3), with error bars representing their SD. The levels of PGF2α formed
were 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 R126
guanidinium 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 reduced
capacity for GSH thiol interaction. A molecule of octyl glucoside bound at the C-domain and low occupancy GSH (∼13%) within the active site of B have been omitted for
clarity (cf. Fig. S3). Polar interactions are shown as dashed lines with distances given in Å.
Brock et al. PNAS | January 26, 2016 | vol. 113 | no. 4 | 973
BIOCHEMISTRY

3. crystal structure indicate the absence of a strong hydrogen-bonding
interaction. Conversely, Arg-126 is observed in a single confor-
mation with an Nη-GSH thiol distance of 3.4 Å. We believe that
this active site geometry also substantiates strong evidence for a
mechanism of GSH thiol activation by an Arg-126 guanidinium
interaction (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 structural
data (13), we wanted to reexamine the functional role of Arg-126
and mutated this residue into both a glutamine and a lysine residue
using site-directed mutagenesis. According to the crystal structure
of mPGES-1 (13), Arg-126 and Asp-49 participate in an inter-
monomeric charge interaction. Therefore, we also mutated the
negatively charged counterpart, Asp-49, into an asparagine residue.
We anticipated that the size and charge conservative mutations of
these residues could serve in probing their role in the enzymatic
mechanism, while minimizing steric and electrostatic repulsion
effects, such as disruption of the monomer interface. Although we
also 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 synthase
activity as described above. For three different purifications of each
isoform, we found that the mutated enzymes did not convert PGH2
into PGE2 above background levels. After preparations of micro-
somal fractions, however, we found that the charge conservative
mutation 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 are
key 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 analysis
of 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 observed
within 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 to
negation of GSH thiol interaction. One possible pathway is show in more detail within Movie S1.
974 | www.pnas.org/cgi/doi/10.1073/pnas.1522891113 Brock et al.

4. in thiolate stabilization if it was already engaged in a stable salt bridge
interaction with Asp-49. Analysis of the relative torsional angles,
however, shows the out-of-plane angle of the Asp-49 carboxylate
relative to the Arg-126 guanidinium to be 44.7° (Fig. 2A). This value
is 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 recent
comprehensive review (31). Therefore, the Asp-49–Arg-126 in-
teraction cannot be classified as the energetically stable, bidentate
interaction of a formal salt bridge and implies that the energetic
barrier for its disruption would be low. This finding provides evidence
for the capacity of these residues to dynamically participate in active
site chemistry and is corroborated by conformational fitting with
qFit (27), which reveals hidden conformations of both residues
depending 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 this
study displayed a promiscuous reductase activity, converting
PGH2 into PGF2α. Notably, the most pronounced activity in this
respect was again observed for microsomal preparations of the
Arg-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 in
the 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 peak
of ∼5 rmsd, ADP = 21.9 Å2
) within the active site (Fig. 2A). By
forming a hydrogen-bonding network from the α-carboxylate
moiety of GSH to its thiol group, it is ideally placed to assist in
deprotonation of the latter during catalysis. The pKa of the
α-carboxylate, in turn, is undoubtedly lowered by the side-on,
out-of-plane interaction with the guanidinium of Arg-38 (torsion
angle 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-sharing
network” that is functionally conserved in all classes of soluble
GSTs for the same purpose (32), and the use of a bridging water
molecule to transfer the thiol proton to the α-carboxylate of
GSH has been shown to be energetically favorable within an
alpha class soluble GST (33). Crucially, density for this water is
absent for the Phenix (34) refined bis-phenyl GSH complex
(PDB ID code 4AL1), in which the relative occupancies to GSH
were refined as 0.87:0.13, respectively. After conformational change
of Asp-49, we hypothesize that Arg-126 can further decrease the
GSH thiol pKa via charge stabilization. The crystallographic
water molecule could then function as the yet-unidentified base
that accepts a proton from GSH during thiolate formation,
concurrently forming a transient hydronium ion or shuttling the
proton to the α-carboxylate. Once formed and stabilized by in-
teraction with Arg-126, we expect attack of GSH thiolate upon
the endoperoxide ring at the C-9 position, resulting in O–O bond
cleavage and proton donation via the hydronium ion. Asp-49,
liberated from its interaction with Arg-126, would now be free to
function as a base within the resulting transition state, facilitating
a decrease of the C-9 proton pKa, and spontaneous decomposition
to yield the product PGE2 and regenerated GSH (Fig. 4). Although
an alternative mechanism in which thiolate would act as a general
base abstracting the C-9 proton has been suggested to be more
energetically favorable in model systems (35, 36), the probability
of either pathway would ultimately be determined by the precise
orientation of substrate relative to cofactor within the enzymatic
active site. Although the apparent ability of the active site mu-
tants characterized here to produce PGF2α via reduction of a
putative sulphenic acid ester intermediate speaks in favor of the
former (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 published
crystal structure of mPGES-1 (PDB ID codes 4AL0 and 4AL1)
(13) to the qFit server (smb.slac.stanford.edu/qFitServer/) (20). This
software automatically samples conformational heterogeneity that is
interpretable by fitting partial occupancy conformational ensembles
into low-level electron density. The CONTACT algorithm was then
used 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 code
4AL0) resulted in a small, but significant, improvement of structure
quality indicators, including the decrease of R/Rfree values from
12.2/13.0% to 11.6/12.8%, respectively. Subsequent analysis of the
structure with CONTACT revealed an extensive network of cor-
related side chain interactions centered upon the short, cytoplasmic
helix separating transmembrane helices I and II that was referred to
by Sjögren et al. (13), and will be hereafter, as the C-domain. Of
most interest is that the network facilitates signal transduction from
residues involved in the recognition of GSH to the active site res-
idue Asp-49. This process could facilitate the disruption of its
interaction with Arg-126, facilitating the latter’s role in thiolate
stabilization on a time scale specific to catalysis. Thr-34 and Leu-69,
located on helices I and II, respectively, make hydrophobic contacts
with the γ-glutamyl moiety of GSH and initiate series of correlated
van 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 the
crystal structure (13) (Fig. 2A). As discussed above, we believe this
active site geometry is strong evidence of a GSH thiol–Arg-126
interaction. Although the pKa of GSH thiol has been measured as
9.42 in solution (37), the dynamic interaction of GSH thiol with
Arg-126, combined with the solvent restricted electrostatics of the
active site, may allow GSH to transiently form thiolate during ca-
talysis via a mechanism of charge redistribution. Specifically, the
hydrogen-bonding network formed by a crystallographic water
molecule between the α-carboxylate and thiol moieties of GSH
may 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 (PDB
ID code 4AL1), in which Arg-126, now with a reduced potential
for 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 and
Discussion.
Brock et al. PNAS | January 26, 2016 | vol. 113 | no. 4 | 975
BIOCHEMISTRY

5. Contact Signaling Is Ligand-Dependent. After an iterative fitting of
alternate conformations with qFit (20), building of N-terminal
residues 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-Å
resolution mPGES-1 complex with bisphenyl–GSH (PDB ID
code 4AL1), with a reduction of R/Rfree values from 16.3/17.2%
to 13.7/16.6%, respectively. Subsequent CONTACT analysis
lacked the extensive signaling network found within the GSH
complex, however, which were instead focused on opposing sides
of the active site. The ensemble structure was found to contain
two networks of four and five residues, respectively, the latter of
which occurs in the cytoplasmic loop between helices III and IV
and contains alternate conformations of Arg-126 (Fig. 3B). This
finding suggests that the activation of dynamic contact networks
in mPGES-1 may be dependent upon the identity of the ligand
bound at the active site. Although the difference in structural
information inherent in the two datasets (1.16 Å cf. 1.95 Å)
should be considered when drawing comparisons between the
qFit-generated ensemble structures, the resolution of the 4AL1
dataset is still significantly higher than the upper limit of 2.1 Å
suggested by the software developers (smb.slac.stanford.edu/
qFitServer/) (20). In addition, we performed a qFit/CONTACT
analysis of high-resolution (1.41–1.52 Å) mPGES-1 inhibitor
complexes recently published (15) (PDB ID codes 4YK5, 4YL0,
4YL1, and 4YL3). These four compounds are also observed to
bind in the intermonomeric active site, making extensive contact
with the C-domain. Although the four inhibitors are varied in
structure and binding modes, they all share a common interaction
with the C-domain and lack the extensive networks found in the
holoenzyme 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/helix
C-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 potential
for signal transduction (Fig. S3). As shown in Fig. 3A and Movie S1,
the dynamic conformations of Lys-41, Arg-40, Leu-39, and His-53
are essential to the transmission of the contact network within the
C-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 structural
ensemble 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 spectrometry
hydrogen/deuterium exchange experiments (38), the authors found
that the greatest differences common to the two most potent inhibitors
were observed in residues 37–54, corresponding to the C-domain.
Conclusions
The combined results of site-directed mutagenesis, functional
assays, structural ensemble, and contact network analysis pre-
sented herein provide strong evidence for a mechanism of PGE2
synthesis by mPGES-1 that features an activation of GSH thio-
late by Arg-126, modulated via its respective interaction with
Asp-49. Furthermore, we show that conformations of the latter
can 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 interaction
during 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 hypothesize
that the active site of mPGES-1 lowers the pKa of GSH thiol
and 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 could
thus be achieved via proton shuffling by the crystallographic
water/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 discussion
within the literature (39).
Materials and Methods
Protein Expression and Purification. Recombinant wild-type (WT) and active-site
mutants of human mPGES-1 were overexpressed in P. pastoris and purified by
Ni-affinity chromatography before exchanging buffer to 0.1 M phosphate
buffer, 0.03% dodecyl maltoside, and 2.5 mM GSH, pH 7.4. Microsomal prep-
arations were prepared via ultracentrifugation of lysed cell supernatant and
homogenization of the microsomal pellets in assay buffer (20 mM Tris·HCl, pH
7.8, 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, please
see SI Materials and Methods.
Enzyme Activity Assay. Conversion of PGH2 to PGE2 by WT or mutated
mPGES-1 were quantified by using GC-MS as described (12). For further
details, 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 for
the quantification of conformational ensembles and functional contact
networks, respectively. Before analysis, the physiological trimer was gener-
ated from the asymmetric unit via crystallographic symmetry using the
program COOT (41). For PDB ID codes 4AL0, 4YL0, 4YL1, 4YL3, and 4YK5, the
coordinates were submitted to the qFit server and refined and prepared for
CONTACT as described (27), by using Phenix-1.9-1692 (34) without manual
intervention. For PDB ID code 4AL1, significant density improvement at the
amino terminus allowed residues 4–9 to be built into density after qFit
conformer fitting and refinement. After a second round of refinement, the
resulting improvement in quality indicators such as the Rfree value were
significant, such that the improved phase estimates were anticipated to
affect 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 CONTACT
analyses 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 assistance
and gratefully acknowledge the late Richard Armstrong, who provided the
GOH GSH analogue. Part of this work was performed at the Karolinska
Institutet Protein Science Facility. Some computations were performed on
resources provided by the Swedish National Infrastructure for Computing
at 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 NovoNordisk
Foundation Grant NNF15CC0018346. J.Z.H. is the recipient of a Distinguished
Professor Award from Karolinska Institutet.
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