Biochem. J. (2011) 440, 13–21 (Printed in Great Britain)
13
doi:10.1042/BJ20110977
Microsomal prostaglandin E synthase-1 exhibits one-third-of-the-sites
reactivity
Shan HE*, Yiran WU*, Daqi YU* and Luhua LAI*†1
*Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering,
Peking University, Beijing 100871, China, and †Center for Theoretical Biology, AAIS, Peking University, Beijing 100871, China
mPGES-1 (microsomal prostaglandin E synthase-1) is a newly
recognized target for the treatment of inflammatory diseases. As
the terminal enzyme of the prostaglandin production pathway,
mPGES-1 inhibition may have a low risk of side effects.
Inhibitors of mPGES-1 have attracted considerable attention as
next-generation anti-inflammatory drugs. However, as mPGES1 is a membrane protein, its enzymatic mechanism remains
to be disclosed fully. We used MD (molecular dynamics)
simulations, mutation analysis, hybrid experiments and co-IP (coimmunoprecipitation) to investigate the conformation transitions
of mPGES-1 during catalysis. mPGES-1 forms a homotrimer with
three substrate-binding sites (pockets). In the MD simulation,
only one substrate molecule could bind to one of the pockets and
form the active complex, suggesting that the mPGES-1 trimer
has only one pocket active at any given time. This one-third-ofthe-sites reactivity enzyme mechanism was verified further by
hybridization experiments and MD simulations. The results of
the present study revealed for the first time a novel one-thirdof-the-sites reactivity enzyme mechanism for mPGES-1, and the
unique substrate-binding pocket in our model constituted an active
conformation that was suitable for further enzymatic mechanism
study and structural-based drug design against mPGES-1.
INTRODUCTION
key residues as gatekeepers for the active site of the enzyme.
Their work is helpful for predicting the substrate-binding site, yet
a comprehensive understanding of the enzymatic structure and
reaction mechanism remains a great challenge.
mPGES-1 is a member of the MAPEG (membrane-associated
proteins in eicosanoid and glutathione metabolism) protein family,
which also includes MGST (microsomal glutathione transferase)1, MGST-2, MGST-3, LTC4 S (leukotriene C4 synthase) and FLAP
(5-lipoxygenase-activating protein) [10]. MGST-1 is the first
member of the protein family for which crystal structures have
been determined [11,12]. On the basis of these crystal structures,
two comparative models of mPGES-1 were built and reported
by two groups: one by Hamza et al. [13], and the other by
Xing et al. [14]. Even before the high-resolution crystal structure
of MGST-1 was published, Zhan and colleagues built the first
model of the substrate-binding domain of mPGES-1 (one pocket)
using an ab initio structure prediction approach [15]. After a 3.2 Å
(1 Å = 0.1 nm) resolution crystal structure of MGST-1 became
available, they built a new structural model of the mPGES-1 trimer
using comparative modelling with MD (molecular dynamics)simulation refinement and experimental validation [13]. On the
basis of this model, several important residues for ligand binding
were identified and their computational predictions about these
residues were supported by the corresponding experimental tests
[16]. However, because both comparative models were mainly
based on the structure of MGST-1, the cofactor GSH in their
models took an extended conformation like that in the MGST-1
structure as pointed out by Pawelzik et al. [9]. Recent advances
in membrane protein studies and structural biology produced
mPGES-1 (microsomal prostaglandin E synthase-1) is a newly
recognized target for the treatment of inflammatory diseases
[1]. NSAIDs (non-steroidal anti-inflammatory drugs) are taken
by more than 30 000 000 people each day around the world.
However, almost all of the currently available NSAIDs have
certain types of side effects (e.g. stomach damage). To gain
more insights into network-based anti-inflammatory drug design,
the dynamic properties of the AA (arachidonic acid) metabolic
network have been studied by us previously [2,3]. In this AA
metabolic network, several enzymes, including leukotriene A4
hydrolase, have been investigated for their enzymatic mechanisms
and inhibitor/activator design [4,5]. mPGES-1 is the terminal
enzyme of the production pathway of prostaglandins (one type of
inflammatory mediator) and is not coupled to any downstream
enzymes in the enzymatic cascade. Constitutive levels of
mPGES-1 are normally low, and it is highly up-regulated by proinflammatory stimuli. Therefore, mPGES-1 is a novel attractive
target with a low risk of side effects [6].
However, the enzymatic reaction mechanism of mPGES-1 has
not been studied much and insufficient information is available
to guide rational inhibitor design. Site-directed mutagenesis was
used to explore the contribution of residues to the enzymatic
reaction [7,8]. Hammarberg et al. [7] reported that the mutation
of Arg126 in mPGES-1 to an alanine residue changed the isomerase
activity to reductase activity. This observation verified Arg126 as
a key catalytic residue. Pawelzik et al. [9] investigated the effects
of different inhibitors on rat/human mPGES-1 and identified
Key words: drug design, enzyme mechanism, inflammation,
membrane-associated proteins in eicosanoid and glutathione
metabolism (MAPEG), microsomal prostaglandin E synthase-1
(mPGES-1), one-third-of-the-sites reactivity.
Abbreviations used: AA, arachidonic acid; co-IP, co-immunoprecipitation; EIA, enzyme immunoassay; FLAP, 5-lipoxygenase-activating protein;
IP, immunoprecipitation; IPTG, isopropyl β-D-thiogalactopyranoside; LTC4 S, leukotriene C4 synthase; MAPEG, membrane-associated proteins in
eicosanoid and glutathione metabolism; MD, molecular dynamics; MGST, microsomal glutathione transferase; mPGES-1, microsomal prostaglandin E
synthase-1; NSAID, non-steroidal anti-inflammatory drug; PGH2 , prostaglandin H2 ; PNP, purine nucleoside phosphorylase; POPC, 1-palmitoyl-2-oleoyl
phosphatidylcholine; RMSD, root mean square deviation; WT, wild type.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
14
S. He and others
an explosion of crystal structures for MAPEG family proteins
including FLAP [17], LTC4 S [18] and mPGES-1 [19]. In the
crystal structures of LTC4 S and mPGES-1, GSH took a U-shaped
conformation. As the residues co-ordinating GSH were highly
conserved in the MAPEG family, the U-shaped GSH-binding
mode should be conserved among MAPEG enzymes. Compared
with the U-shaped conformation, the extended conformation of
GSH in MGST-1 might correspond to a structurally distorted form
[20]. Therefore the problem of MGST-1 crystal structure might
influence the comparative modelling of mPGES-1 which used the
structure of MGST-1 as the template structure, especially for
the GSH-binding site.
Jegerschöld et al. [19] determined the structure of mPGES-1 by
electron crystallography to a resolution of 3.5 Å. The mPGES-1
molecule in this structure took a closed inactive conformation
that was not accessible by the substrate PGH2 (prostaglandin
H2 ). As the crystal structure of LTC4 S was solved with an open
conformation to a resolution of 2.15 Å, by comparing these two
structures they speculated that a conformation change might occur
during binding and turnover of the substrate. Enlightened by
this idea, we attempted to build an open conformation structural
model of mPGES-1 and used MD simulations to investigate the
conformation transition between the open and closed state. During
the MD simulation, only one substrate molecule was observed
to bind to the pocket and form an active complex, suggesting
that the mPGES-1 trimer might only have one pocket active
at any given time. This one-third-of-the-sites reactivity enzyme
mechanism was further supported by hybridization experiments
and prolonged MD simulations.
EXPERIMENTAL
Materials
The plasmid DNA harbouring the full-length cDNA of the
PIG12 gene was obtained from Addgene [Addgene plasmid
16506: pBK-CMV Pig12 (CMV is cytomegalovirus)]. PGH2 ,
anti-(mPGES-1) monoclonal antibody, mPGES-1 Western blotready control and the PGE2 EIA (enzyme immunoassay) kit
were purchased from Cayman Chemical. POPC (1-palmitoyl2-oleoyl phosphatidylcholine) was obtained from Avanti Polar
Lipids. Immunoprecipitation Kit Dynabeads® Protein G and
DynaMagTM -2 magnets were purchased from Invitrogen. An antiHis6 -tag antibody and a rabbit polyclonal secondary antibody
against mouse IgG were purchased from Abcam. Other reagents
were obtained from Sigma–Aldrich unless indicated otherwise.
Cloning and plasmid construction
The coding region of mPGES-1 from the plasmid DNA was
PCR-amplified using the primer pair 5 -CGTGTACATATGCCTGCCCACAGCCTG-3 and 5 -CTAGAATTCACAGGTGGCGGGCCGCTTC-3 . The NdeI/EcoRI fragment was ligated into a
pET30a ( + ) vector. Three CGG codons of mPGES-1 encoding
the Arg40 , Arg73 and Arg122 were changed to CGT codons using a
Muta-direct kit (SBS) to improve mPGES-1 expression [21]. For
further co-IP (co-immunoprecipitation) analysis, we also constructed His–mPGES by ligating the NdeI/EcoRI fragment into a
pET28a ( + ) vector. After ligation, an extended His6 -tag (MGSSHHHHHHSSGLVPRGSH) was attached to the N-terminus of
mPGES-1. Therefore the protein bands of the fusion protein
His–mPGES (172 amino acids, theoretical molecular mass of
18 kDa) and mPGES-1 (152 amino acids, theoretical molecular
mass of 16 kDa) could be separated by SDS/PAGE. The proteincoding region of the resulting expression vector was sequenced
for verification.
c The Authors Journal compilation c 2011 Biochemical Society
Protein expression and microsome preparation
Human recombinant mPGES-1 was overexpressed in the Rosetta
(DE3) strain of Escherichia coli. Recombinant cells were
cultivated at 37 ◦ C in LB (Luria–Bertani) medium containing
30 μg/ml kanamycin and 34 μg/ml chloramphenicol until an
attenuance value of D600 = 1.0 was reached. Protein expression
was induced by the addition of IPTG (isopropyl β-Dthiogalactopyranoside) to a final concentration of 1 mM, and the
cells were grown for a further 12 h at 25 ◦ C. Cells were harvested
by centrifugation at 5000 g for 20 min at 4 ◦ C.
The cell pellet from a 1 litre culture was resuspended in lysis
buffer [1 mM EDTA, 1 mM PMSF and 10 % (v/v) glycerol
(pH 7.4)] and lysed by ultrasonication (350 W, 99× 3 s bursts
with 5 s rest between each burst) until homogeneous. Insoluble
material was separated by centrifugation (12 000 g for 30 min
at 4 ◦ C), and the supernatant was then ultracentrifuged at
52 000 rev./min, for 1 h with a Hitachi rotor (S58A-0026). The
membrane pellet was washed in activity assay buffer [2.5 mM
GSH, 0.1 M potassium phosphate buffer (pH 7.4) and 1 % (v/v)
glycerol] once and then resuspended in 2 ml of solubilizing buffer
[2.5 % (w/v) POPC in activity assay buffer]. The total membrane
protein concentrations were determined using a bicinchoninic
acid protein assay kit (Biomed Laboratories) with BSA as a
standard. According to the concentration, the microsome samples
were diluted to a final concentration of 10 mg/ml. WT (wild-type)
and mutant mPGES-1 microsome samples and negative control
(microsome prepared with Rosetta cells without IPTG induction)
were all prepared in this manner.
Enzymatic activity assay
mPGES-1 activity was measured by quantifying the conversion
of PGH2 into PGE2 according to a method reported previously
[22]. The microsome was diluted in activity assay buffer to the
desired concentration. Because the substrate PGH2 was labile, it
was always kept on dry ice until just prior use. Prior to incubation,
both the substrate and samples were transferred on to ice for 2 min
for temperature equilibrium. PGH2 was added to each well of a
96-well plate, and the reaction was started by adding 100 μl of the
samples. After reacting at 4 ◦ C for 1 min, the reaction was terminated by adding 150 μl of stop solution (50 mM FeCl2 and 100 mM
citric acid) to lower the pH to 3. PGE2 in the reaction mixture was
quantified using the PGE2 EIA kit (Cayman Chemical).
Site-directed mutagenesis and activity comparison between
the WT and mutants
Mutagenesis was performed using a Muta-direct kit (SBS). The
plasmid pET30a ( + ) containing WT mPGES-1 was mutated
to obtain the following mutants: R38A, R38S, H72A, N74A,
E77A, R110A, Y117A, Y117F and R126A. The plasmid pET28a
( + ) containing His–mPGES was mutated to obtain His–
R126A. All DNA sequences of the mutants were verified by
DNA sequencing. The protein expression and activity assay
of mutants were performed as described for WT. As the expression levels of mutants and WT might be different, the mPGES1 content in the microsome samples might be different, which
would affect the comparison of their activities. To eliminate the
influence of enzyme concentration, we divided the enzymatic
activity by the mPGES-1 content in the sample (determined
by Western blot analysis, see Supplementary Figure S3 and
Supplementary Table S3 for quantification details at http://www.
BiochemJ.org/bj/440/bj4400013add.htm) to obtain the unit
activity. Then, for comparison, the unit activities of mutants were
represented as the relative activity compared with that of the WT.
One-third-of-the-sites reactivity of mPGES-1
Hybridization experiment
Microsome samples with a constant molar ratio (10:1) of
R126A/WT were mixed for various periods to investigate the
mixing process. The incubation temperature was 25 ◦ C, with
gentle shaking at 500 rev./min. To reach the 10:1 molar ratio,
we analysed the mPGES-1 expression level in the R126A and
WT samples by Western blotting (see Supplementary Figure
S3C lanes 4–5 and Figure 3C lanes 1 and 3). As their expression
levels were very similar, 50 μl of WT and 500 μl of R126A
samples were mixed directly. Then, maintaining a constant
incubation time (28 h), different molar ratios of R126A/WT were
mixed in the same manner while keeping the amount of WT
constant [23]. A control experiment using WT and a negative
control was performed in the same way. The activity of the final
enzyme mixture was determined using the PGE2 EIA kit.
Co-IP
Prior to co-IP, microsome samples with equal amounts of WT
and His–R126A were mixed at 25 ◦ C with rotation for 28 h. CoIP was performed using an immunoprecipitation kit (Invitrogen)
according to the manufacturer’s protocol. Briefly, Dynabeads
Protein G (1.5 mg) were incubated with 3 μg of antibody [using
an anti-His6 -tag antibody for anti-His IP (immunoprecipitation)
and an anti-mPGES-1 antibody for anti-mPGES IP] for 20 min
with rotation at 25 ◦ C. The antibody-bound Dynabeads were then
gently washed and microsome sample mixture (typically 500 μl)
was added. After 1 h of incubation, the supernatant was removed
(collected for further Western blot analysis) and the Dynabeads
were washed three times. The Dynabeads were then resuspended
in 20 μl of elution buffer and 20 μl of 2 × SDS loading buffer,
followed by heating at 70 ◦ C for 10 min.
The immunoprecipitated proteins in the eluent were resolved by
15 % Tricine-SDS/PAGE and transferred on to PVDF membranes
(Millipore) for Western blot analysis. Primary antibody
(1:500 anti-mPGES-1 antibody for anti-mPGES-1 Western blot
and 1:1000 anti-His6 -tag antibody for anti-His Western blot) and
rabbit polyclonal secondary antibody against mouse IgG were
used together with the BCIP (5-bromo-4-chloroindol-3-yl phosphate)/NBT (Nitro Blue Tetrazolium) Color Development Substrate (Promega) to visualize the proteins. A control experiment
using a WT and R126A mixture and a parallel experiment using
R126A and His–WT mixture were performed in the same manner.
Construction of the mPGES-1 open conformation structural model
with PGH2 and structure refinement using MD simulation
As mPGES-1 was solved in a closed conformation (PDB code
3DWW) and LTC4 S was solved with an open conformation (PDB
code 2UUH), we compared and superimposed these two structures
using the program PyMOL (http://www.pymol.org; Figure 2). The
main difference lay in the bending direction of the cytoplasmic
half of helix 1 and helix 2 (active region) over a hinge fixed by
the Lys26 –Asp75 salt bridge, as Jegerschöld et al. [19] suggested.
Therefore we manually bent this region over the hinge with
reference to the corresponding helix in LTC4 S to ‘open’ the
substrate-binding pockets. As mPGES-1 is a trimer that has three
possible substrate-binding sites, all three sites were ‘opened’ using
the same procedure initially. As the structure was determined
without the presence of substrate, we docked the substrate PGH2
into the binding site. Prior to the molecular docking of PGH2 , the
tentative open conformation structure was optimized by energy
minimization and MD simulation with the protein embedded into
the POPC phospholipid bilayer surrounded by water molecules
using the GROMACS software package version 4.0.5 with the
15
GROMOS96 43a1 Forcefield [25]. The final system contained
three protein chains, three GSH moieties, 98 POPC moieties,
3877 water molecules and 21 Cl − ions (a total of 21191 atoms in
a 63 Å × 63 Å × 76 Å simulation box). All simulations were
run under constant pressure (1 bar) and temperature (310 K)
using the Parrinello–Rahman method [26] for pressure control
and the Nose–Hoover method [27] for temperature control. A
12-Å cut-off was used for the non-bonded interactions, and
long-distance electrostatic interactions were treated using the
particle mesh Ewald method [28]. The bonds with hydrogen
atoms were constrained by the LINCS algorithm [29], and the
time step was set to 1.0 fs. Harmonic restraint with a stiffness
of 100 kJ · mol − 1 · nm − 2 was applied on the α-carbon atoms of
proteins to avoid unwanted pocket closing.
PGH2 was then docked to the three pockets separately by
AutoDock 4 using a genetic algorithm with the following
parameters: ga_num_evals = 10000000, ga_pop_size = 300 and
ga_run = 100. Arg116 , Tyr120 , Gln124 and Gln130 were treated as
flexible residues to aid the docking. The final conformation
of PGH2 was selected with consideration of binding energy,
clustering, and proper protein–substrate interactions. This
mPGES-1–PGH2 complex structure was further optimized by
MD simulation with progressively restraint reduction until full
relaxation was achieved. MD simulations of 100 ns (notated
as refining-MD simulation) were required to obtain a stable
open conformation complex structural model of mPGES-1
(notated as open conformation, see Supplementary Table S1 and
Supplementary Figure S1 at http://www.BiochemJ.org/bj/440/
bj4400013add.htm for MD simulation details).
Construction of pseudo-all open structure model of mPGES-1 and
prolonged MD simulations
To elucidate the conformation transition mechanism and confirm
the unsymmetrical structure of the trimer, we also built a pseudoall open structure and studied its conformational changes during
MD simulation. The all open structure was built using the second
monomer of mPGES-1 with GSH (the open conformation)
to generate a symmetric trimer. After repacking the enzyme to
the membrane environment and energy minimization, a 50 ns
fully relaxed MD simulation was performed to investigate the
conformation transition elicited by this change (notated
as simulation A). The energy minimization steps and MD
simulation parameters were the same as the modelling section
(see above). The reverse transition from the all closed structure
(PDB code 3DWW) was also studied by 50 ns fully relaxed MD
simulations in the membrane environment (notated as simulation
B). Simulation A and B were performed in three independent
runs, and at least two trajectories exhibited similar results. The
final structure after MD simulation (after-MD structure) was
taken directly as the last frame.
Inhibitor docking
We collected mPGES-1 inhibitors that had available IC50 values
based on the cell-free microsome assay in the literature (a
total of 117 molecules belonging to 19 different types, see
Supplementary Table S2 at http://www.BiochemJ.org/bj/440/
bj4400013add.htm). The inhibitors were docked to the substratebinding pocket using the program AutoDock 4 (genetic
algorithm with parameters of: ga_num_evals = 50000000,
ga_pop_size = 300 and ga_run = 50). Docked structures were
clustered on the basis of the RMSD (root mean square deviation)
value, and only the structure with the lowest energy in the largest
cluster was selected as the best docking result. The correlation
c The Authors Journal compilation c 2011 Biochemical Society
16
Figure 1
S. He and others
Open conformation structural model of mPGES-1
(A) Final model of mPGES-1 (the three monomers are coloured green, magenta and cyan) with cofactor GSH (yellow) and substrate PGH2 (blue). The distances between the oxygen atoms of PGH2
and α-carbon atoms of Arg73 are labelled to represent the extent to which the substrate entered the pocket. (B) The PGH2 –Arg73 distance variation during the MD simulation. (C–E) Closer view of
pocket-1, -2 and -3. The PGH2 –GSH and PGH2 –Arg126 distances were labelled to illustrate the substrate-binding mode. (F) Hypothetical enzyme reaction mechanism involves an attack of the GSH
thiolate and protonation of Arg126 [19].
between experimentally determined IC50 values and calculated
K d values was analysed.
RESULTS
Construction of the open conformation structural model of
mPGES-1 with PGH2 and MD simulation refinement
mPGES-1 forms a homotrimer with three substrate-binding
pockets and each of the pockets is buried at the interface of two
adjacent monomers. mPGES-1 catalyses the conversion of PGH2
into PGE2 and requires GSH as an essential cofactor for activity.
The mPGES-1 structure (PDB code 3DWW) was determined in
complex with GSH, but without substrate [19]. As there were no
reports about the binding ratio of PGH2 to the enzyme trimer,
we tentatively docked PGH2 in all three pockets. Surprisingly,
these three PGH2 molecules moved differently during the MD
simulation (Figure 1). We numbered the pockets clockwise as
pocket-1, pocket-2 and pocket-3 for clarity. To represent the extent to which the substrate entered the pocket, we used the
c The Authors Journal compilation c 2011 Biochemical Society
distance between the oxygen atom of PGH2 and α-carbon atom
of Arg73 because Arg73 was located at the bottom of the pocket
and positioned on the same horizontal plane as PGH2 . In pocket-1
and pocket-3, the distance between PGH2 and pocket grew from
14 Å to more than 17 Å, whereas in pocket-2 the distance was
reduced from 14 Å to less than 12 Å. Therefore, during the
refining-MD simulation, only the substrate near pocket-2 entered
the pocket to form an active complex, whereas the other substrates
moved away from the pocket. This implied that the mPGES-1
trimer might have only one pocket active at any given time. The
cofactor GSH remained tightly bound only in pocket-2. Recent
studies of a homologous protein MGST-1 indicated that its three
pockets had different affinities for GSH with one high-affinity
(K d = 20 μM) and two low-affinity (K d = 2.5 mM) sites [30]. Our
MD simulation results agree well with these experimental data.
In pocket-2, the binding mode of PGH2 was suitable for
enzymatic reaction. The endoperoxide bridge of PGH2 formed a
hydrogen bond with the guanidine group of Arg126 (3.4 Å) and was
close to the thiol group of GSH (4.4 Å). This was consistent with
the reaction mechanism proposed by Jegerschöld et al. [19], which
One-third-of-the-sites reactivity of mPGES-1
Figure 2
Important residues validated by mutagenesis
(A) Important residues in pocket-2 identified by our model that formed hydrogen bonds with
GSH. (B) Relative enzymatic activities of mPGES-1 WT and mutants. The WT activity was set as
100 %. The enzymatic activity was measured by quantifying the conversion of PGH2 into PGE2
as described in the Experimental section. Results are means +
− S.E.M. (n = 3).
involved the participation of GSH and Arg126 . Tyr117 and Tyr130
were also close to the endoperoxide bridge of PGH2 (within 5 Å).
Validation of important residues identified in the model
by mutagenesis
In pocket-2, seven residues (Arg38 , His72 , Asn74 , Glu77 , Arg110 ,
Arg126 and Tyr117 ) formed hydrogen bonds with GSH or
PGH2 , which may play important roles in enzyme catalysis.
Although there were mutation data published for six of them
(except for Arg38 ) by different research groups [7,8,19], for
comparison we mutated all seven residues and compared their
activities (see Supplementary Figure S2 at http://www.BiochemJ.
org/bj/440/bj4400013add.htm for a comparison between the
results of the present study and the published data). The enzymatic
activity of all mutants decreased dramatically to less than 30 %,
which verified the importance of these residues (Figure 2). We also
examined the less important residues (defined as residues that after
their mutation mPGES- activity remained above 50 %) identified
by published mutation data [7,8,15,19]. In pocket-2 of our model,
the unimportant residues were not located near the substratebinding pocket, in good agreement with the experimental results
(see Supplementary Figure S2 for details).
In summary, the conformation of pocket-2 can explain the
reaction mechanism well, and the catalytic residue Arg126 , and
the important and unimportant residues for enzyme reaction can
be predicted correctly. Thus the structure of pocket-2 provides a
good representation of the substrate-binding conformation.
Hybridization experiment and co-IP
To further verify the one-third-of-the-sites reactivity mechanism,
we performed hybridization experiments by mixing the inactive
R126A mutant (M) with the active WT enzyme (W) to produce
17
hybrid trimers. Considering Arg126 was located far from the
subunit interface and therefore was unlikely to influence
the hybrid trimer formation, we selected the R126A mutant as the
inactive mutant. There were four possible species (WWW, WWM,
WMM and MMM) in the enzyme mixture. For the mPGES-1
trimer, there were three possible enzymatic mechanisms. (i)
Mechanism 1: all-sites reactivity (only WWW is active). In this
situation, if we add the mutant to the WT sample, the activity of the
enzyme mixture will decrease as more hybrids will be formed. (ii)
Mechanism 2: one-third-of-the-sites reactivity (WWW, WWM
and WMM are all active). One-third-of-the-sites reactivity means
that the mPGES-1 trimer needs at least one active monomer to
be active, and therefore WWW and both hybrids (WWM and
WMM) should be active. In this situation, adding increasing
amount of the mutant to the WT would result in the increasing
formation of active hybrids, and the total activity would increase.
The maximum activity of the enzyme mixture would be three
times that of the WT as one WWW could recombine to form
three WMM hybrids; (iii) mechanism 3, two-thirds-of-the-sites
reactivity (both WWW and WWM are active). In this situation, the
activity of the enzyme mixture would initially increase and then
decrease when a higher concentration of the mutant is mixed with
the WT. This is because during the process of mixing, the WT and
mutant first form the active hybrid WWM, giving an increase in
the total activity. When more inactive mutant is added the WWM
hybrid will be transformed into the inactive hybrid WMM and the
activity of the mixture would decrease. The turning point should
occur when the mutant and WT are in an equal molar ratio. At this
point, the total concentration of active WWW and WWM will
be the same as those of the inactive WMM and MMM. Increasing
the concentration of the mutant after this point would result in the
formation of additional inactive species, thereby reducing
the activity of the enzyme.
On the basis of the differences between these three
possible enzymatic mechanisms, we designed two hybridization
experiments (Figure 3). First, we mixed samples containing
a constant molar ratio (10:1) of R126A/WT for various time
periods to investigate the mixing process. Figure 3(A) shows
that the experimentally measured enzymatic activity increased
with incubation time and reached equilibrium at approximately
28 h. This result excluded the possibility of mechanism 1
and supported mechanism 2. We then kept the incubation
time at 28 h and mixed different molar ratios of R126A/WT
(keeping the concentration of WT constant). The experimental
enzymatic activity increased as the R126A/WT molar ratio
increased (even after R126A/WT = 1) and increased by a factor of
approximately three when R126A/WT = 10. This result excluded
the possibility of mechanism 3 and supported mechanism 2. We
also performed a control experiment by adding the negative
control to the WT, upon which no increase in activity was
observed. Therefore the activity increases observed in the two
experiments should be caused by the addition of R126A, and
the experimental results supported mechanism 2 (one-thirdof-the-sites reactivity). For the second experiment, we also
calculated the theoretical activity curve for the three possible
enzymatic mechanisms, and the experimental value was in good
agreement with the one-third-of-the-sites reactivity mechanism
(see Supplementary Figure S4 at http://www.BiochemJ.org/
bj/440/bj4400013add.htm for a detailed analysis).
We have assumed that the mutant and WT can form hybrid
trimers when mixed in solution. To verify the hybrid trimer
formation, we further conducted co-IP analysis (Figure 3C). To
distinguish the mutant and WT monomer, we attached an extended
His6 tag (MGSSHHHHHHSSGLVPRGSH) to the N-terminus
of R126A. The protein bands of the fusion protein His–R126A
c The Authors Journal compilation c 2011 Biochemical Society
18
Figure 3
S. He and others
Hybridization experiments and co-IP
(A) Microsome samples with a constant molar ratio (10:1) of R126A/WT were mixed for various periods to investigate the mixing process. A control experiment was also performed using a WT and
negative control (NC; microsome prepared with Rosetta cells without IPTG induction) mixture. The enzymatic activity was measured by quantifying the conversion of PGH2 into PGE2 as described in
the Experimental section. The activity of the enzyme mixture was represented by the relative activity (the starting point of the WT + R126A activity curve when the incubation time was 0 h was set as
100 %). Results are means +
− S.E.M. (n = 3). (B) Microsome samples with different molar ratios of R126A/WT were mixed for 28 h before the activity assay. A control experiment was also performed
using a WT and negative control mixture. The activity of the enzyme mixture was represented by the relative activity (the starting point of the WT + R126A activity curve when the R126A/WT molar
ratio equalled 0 was set as 100 %). Results are means +
− S.E.M. (n = 3). (C) Samples containing equal amounts of WT (theoretical molecular mass of 16 kDa) and His–R126A (theoretical molecular
mass of 18 kDa) (or parallel experiments using R126A and His–WT samples) were mixed for 28 h and then immunoprecipitated with anti-mPGES-1 antibody (mPGES IP*) or anti-His6 -tag antibody
(His IP). Both the immunoprecipitated proteins (IP) and the supernatant (S) were analysed. The samples were resolved on two identical gels, which were analysed by Western blotting (WB) using
the anti-mPGES-1 antibody (top panel, anti-mPGES-1 Western blot) or anti-His6 -tag antibody (bottom panel, anti-His6 tag Western blot). The additional band detected above 18 kDa in the anti-His6
tag Western blot was a non-specific band. Lanes 1 and 2 are WT and His–R126A microsome samples respectively. Lanes 5–7 are co-IP analysis of WT and His–R126A mixture. Lanes 3 and 4 and
8–10 are a parallel experiment using R126A and His–WT mixture. A representative result of three independent experiments is shown. A control experiment using WT and R126A mixture was also
performed (see Supplementary Figure S3C at http://www.BiochemJ.org/bj/440/bj4400013add.htm for details). Molecular mass in kDa is given on the left-hand side.
(172 amino acids, theoretical molecular mass of 18 kDa) and WT
(152 amino acids, theoretical molecular mass of 16 kDa) can be
separated by SDS/PAGE. We also tested the enzymatic activities
of WT, His–WT, R126A and His–R126A. The attachment of
the His6 tag had no effect on the enzymatic activity (WT and
His–WT had comparable activity and both R126A and His–
R126A had no activity; see Supplementary Figure S3B for
details). IP of the WT and His–R126A mixture with the antimPGES-1 antibody pulled down all detectable mPGES-1 moieties
(both WT and His–R126A) from the supernatant, which can
be used to represent the total amount of WT and His–R126A
in the mixture. In comparison, IP with the His6 -tag antibody
pulled down all His–R126A and a large percentage of WT from
the supernatant. Because IP of the WT and R126A mixture
with the anti-His6 -tag antibody could not pull down WT or
R126A from the supernatant (see Supplementary Figure S3C
for control experiment details), the Western blot analysis using
the anti-His6 -tag antibody could not detect WT or R126A bands
(Figure 3C). There was no cross-reaction between the WT
and anti-His6 -tag antibody. Therefore pull down of the WT by
the anti-His6 -tag antibody should be mediated by its interaction
with His–R126A, indicating hybrid trimer formation between the
mutant and WT. We also performed a parallel experiment using
R126A and His–WT mixture for co-IP analysis. The anti-His6 tag antibody pulled down both His–WT and R126A from the
supernatant. This result further supported the formation of hybrid
trimers of mutant and WT.
c The Authors Journal compilation c 2011 Biochemical Society
In summary, the co-IP analysis confirmed that the mutant and
WT can form hybrid trimers in solution, and the two hybridization
experiments showed that the activity of the mixture increased
with increasing hybrid formation. Therefore the results provided
evidence for the one-third-of-the-sites reactivity mechanism.
MD simulation of conformation transition process
We performed simulation A (from pseudo-all open structure) and
simulation B (from all closed structure) to further elucidate the
conformation transition mechanism and verify the unsymmetrical
structure of the trimer (Figure 4). To represent the extent to
which the substrate-binding pocket opened, we used the distance
between the α-carbon atoms of Leu39 and Arg126 , as they were
located at the termini of helices 1 and 4 in an approximately
horizontal direction. The three pockets in the after-MD structure
were named pocket-1 , pocket-2 and pocket-3 for simulation A
and pocket-1 , pocket-2 and pocket-3 for simulation B.
During simulation A, two pockets closed quickly and
transformed to the closed conformation, with only pocket-3
remaining open. This result suggested that there was a constraint
force between the monomers and simultaneous opening of the
three pockets was not permitted. Figures 4(D) and 4(E) illustrate
the molecular surface representations of the conformations. In the
open conformation, the pocket was open, and the cofactor GSH
could be observed from outside of the binding pocket. After
the MD simulation, the distances between the two monomer
One-third-of-the-sites reactivity of mPGES-1
Figure 4
19
Simulation A (from pseudo-all open structure) and simulation B (from all closed structure)
(A) Superimposition of mPGES-1 (magenta and green) and LTC4 S (white). The main difference lay in the bending direction of the cytoplasmic half of helix 1 and helix 2 (active region light magenta)
over a hinge fixed by the Lys26 –Asp75 salt bridge (arrow). The RMSD fluctuation of Cα atoms of pocket-2 during simulation A is shown on the right. The maximum (with a light magenta bar below, the
bar is coloured following the left-hand panel) corresponds with the active region. (B and C) Simulation A–simulation B. Closing path from open (white) to closed (magenta and green) and opening
path from closed (white) to open (magenta and green) suggested by the simulation and the Leu39 –Arg126 distance variation during the simulation. The distance between the α-carbon atoms of Leu39
and Arg126 was labelled to illustrate the extent of opening of the substrate-binding pocket. The cofactor GSH is coloured yellow, and the after-MD structure is shown in pink. (D and E) Simulation
A–simulation B. Molecular surface images of the mPGES-1 trimer before (top panel) and after (bottom panel) the MD simulations. The three pockets are numbered from left to right.
pairs decreased and the monomers entirely surrounded GSH
making it inaccessible to the substrate. The MD simulation
also demonstrated how the conformation transition occurred. As
Jegerschöld et al. [19] solved the crystal structure of mPGES-1
in a closed conformation, they speculated that it could transform
to the open conformation by bending the cytoplasmic halves of
helix 1 and helix 2 (active region) over a hinge fixed by the Lys26 –
Asp75 salt bridge. The maximal RMSD fluctuation of Cα during
the simulation (using pocket-2 as an example, with the other two
pockets showing similar results) corresponded with this region,
supporting the proposed transformation mode.
Simulation B indicated that only one pocket (pocket-2 ) opened
during the MD simulation, whereas the other two pockets
remained closed. As our model was constructed by manually
bending helix 1 to open the pocket, this simulation validated
that our tentative open conformation was transformable from the
experimentally determined closed conformation. Furthermore, it
suggested that the mPGES-1 trimer tended to open only one
pocket at any given time, supporting the one-third-of-the-sites
reactivity mechanism.
Superimposition of the after-MD structures and open/closed
conformation revealed that, whereas in simulation A the pockets
fully transformed to the closed conformation (the Leu39 –Arg126
distance was reduced from 13 Å to 8 Å), in simulation B the
pocket was only partially open (the Leu39 –Arg126 distance
grew from 8 Å to 12 Å). To reach the fully open conformation,
we attempted to extend simulation B to 100 ns (simulation B + ) or
manually placed a molecule of PGH2 near one pocket (simulation
C) (see Supplementary Figure S3 for details). However, the
conformation transition still could not be completed and PGH2
could not be docked in the partially open pocket. Recently,
Nury et al. [31] performed 1 μs MD simulations of an ion
channel in a fully hydrated membrane environment, and the
conformation transition was incomplete at the end of simulation.
They speculated that the conformation transition mediated by
helix bending was a slow process, especially in the lipid
bilayer environment. Therefore although the ideal method of
building an open conformation structural model of mPGES1
may be extending simulation B until a fully open conformation
was obtained, its implementation was limited to computational
demands. To strike a balance between speed and accuracy, we
used a manually generated initial configuration combined with a
refining-MD simulation strategy to build the open conformation
structural model of mPGES-1, as described in the Experimental
section. As our model was well supported by the experimental
results, this strategy may also be useful for other slow-process
studies.
Validation of the pocket-2 structure by the molecular docking
of inhibitors
In our model, pocket-2 provided a good representation of the
active substrate-binding conformation and therefore should be
the target conformation of mPGES-1 inhibitors. We performed
c The Authors Journal compilation c 2011 Biochemical Society
20
Figure 5
S. He and others
Molecular docking of inhibitors
Published mPGES-1 inhibitors (117 molecules belonging to 19 different types) were docked to
the active substrate-binding pocket-2 using the program AutoDock 4. Correlation analysis was
performed between experimental pIC50 and calculated pK d values. The correlation coefficient R
was 0.74.
a rigorous test of this model by docking all of the collected
inhibitors (117 molecules belonging to 19 different types, see
Supplementary Table S2) in this pocket and analysed the
correlation between the experimental pIC50 and predicted pK d
values (Figure 5). The correlation coefficient R reached 0.74,
which was good for docking studies based on a large set of
inhibitors. This result further supported pocket-2 as an active
substrate-binding pocket.
DISCUSSION
In biological systems, proteins often self-associate to form dimers
or higher-order oligomers [32]. Protein oligomerization is a
key factor in regulating the biological functions of proteins,
such as enzymes, ion channels, receptors and transcription
factors. In previous studies, the regulatory roles of protein
dimerization were extensively explored [33–35]; however, much
fewer reports concerning trimeric proteins are known. The most
difficult problem is the relative paucity of experimental data
for trimeric proteins. A search of the Brenda enzyme database
(a comprehensive enzyme information system containing 5117
different enzymes, http://www.brenda-enzymes.org) indicates
that among 611 human enzymes with PDB entries, 248 enzymes
are dimers, whereas only 27 are trimers.
The results of the present study have proved for the
first time that the mPGES-1 trimer has one-third-of-thesites reactivity. Recent studies of MGST-1 indicate that its
three pockets have different affinities for GSH and exhibit
similar one-third-of-the-sites reactivity [30]. Therefore this
enzymatic mechanism might be conserved in the MAPEG
family. For the one-third-of-the-sites reactivity of mPGES-1,
there are two possible ways of activity regulation: mPGES-1
undergoes breathing motion and randomly opens one pocket
to be active (random way) or a sequential catalytic process
similar to that of ATP synthase occurs. Although mPGES-1
shares a similar 3-fold structure and one-third-of-the-sites
reactivity with ATP synthase, the structure of ATP synthase is
much more complex, which consists of at least 22 subunits and is
powered by a proton gradient, whether this molecular motor can
be accomplished by a simple homotrimer structure remains an
open question [36]. Furthermore, the sequential catalytic process
implies that inactivating one site will lead to complete inactivation
[37]. Our hybridization experiment revealed that hybrid species
WMM and WWM had comparable activity with that of the WT
enzyme, which indicated that the random way was more plausible.
However, unlike the sequential catalytic process for which
it is easy to explain why the mPGES-1 trimer has only onethird-of-the-sites reactivity, if mPGES-1 randomly opens one
c The Authors Journal compilation c 2011 Biochemical Society
Figure 6
Possible mechanism for mPGES-1 one-third-of-the-sites activity
(A) Different motion mode for the single pocket (top panel) and associated three pockets (bottom
panel). (B) Annular interaction network formed by the close packing of helices 1 and 2 (shown
in cylinder) and cofactor GSH (molecular surface representation). (C) Central cavity (black dot)
in the mPGES-1 trimer calculated using the program Cavity [40].
pocket to be active, why does it require oligomerization to
form homotrimers? In principle, one-third-of-the-sites reactivity
lowers the capacity of enzyme and as a consequence might
not be favoured by evolution. On the basis of the results
of the present study and those of previous studies, we
propose that in mPGES-1 the active pocket needs inactive
pockets for both functional and structural purposes. mPGES-1
relies on an open-to-closed conformation shift to perform its
enzymatic reaction; a single pocket may fluctuate in three
directions and this flexibility hinders efficient transition motion.
A simple solution to this is associating three pockets together to
form a homotrimer (Figure 6). Griffin et al. [38] reported that for
dihydrodipicolinate synthase, the homotetrameric structure was
more rigid than the dimeric form, thus reducing excessive relative
motions and improving the enzymatic activity. In mPGES-1,
the two inactive pockets may also sterically stabilize the active
site and only permit the motion responsible for the conformation
transition. This steric stabilization effect may be fulfilled by the
close packing of helices 1 and 2 and cofactor GSH, forming an
annular interaction network (Figure 6B). Furthermore, mPGES-1
may form homotrimers for structural purposes. In addition to the
substrate-binding pocket, the three subunits of mPGES-1 form a
central cavity (Figure 6C). Residues located inside the cavity are
mainly polar residues such as arginine and glutamic acid. The
folding of α-helical membrane proteins has been conceptualized
by a two-stage model [39]. The first stage is membrane insertion
and secondary structure formation, and the second stage is helix–
helix association. Polar residues located in the interface of the
subunits can form interhelical hydrogen bonds in the second
stage that contribute to protein stability substantially. We believe
that these two effects explain the enzymatic mechanism of
the mPGES-1 trimer. However, other explanations may also be
possible, and further experimental evidence is needed to unravel
the dynamics of the entire reaction process.
In conclusion, we have identified a novel one-third-of-thesites reactivity mechanism for the mPGES-1 trimer. An open
conformation structural model of mPGES-1 was also built, which
behaved well in known inhibitor docking and structure–activity
relationship studies. This active conformation model of mPGES-1
can be further used in novel inhibitor discovery and other related
studies.
AUTHOR CONTRIBUTION
Shan He performed the experiments and computational work, analysed data and wrote the
paper; Yiran Wu and Daqi Yu guided and optimized the computational work and edited
the paper prior to submission; Luhua Lai, the scientific supervisor, initiated the study and
conceived the research, edited and approved the final paper.
One-third-of-the-sites reactivity of mPGES-1
ACKNOWLEDGEMENTS
We thank the Shanghai Supercomputer Center of China for providing highperformance computing resources, and Changsheng Zhang and Yaxia Yuan for helpful
discussions.
FUNDING
This work was supported, in part, by the Ministry of Science and Technology of China
and the National Natural Science Foundation of China [grant numbers 10721403 and
90913021].
REFERENCES
1 Samuelsson, B., Morgenstern, R. and Jakobsson, P. J. (2007) Membrane prostaglandin E
synthase-1: a novel therapeutic target. Pharmacol. Rev. 59, 207–224
2 Yang, K., Ma, W. Z., Liang, H. H., Qi, O. Y., Tang, C. and Lai, L. H. (2007) Dynamic
simulations on the arachidonic acid metabolic network. PLoS Comput. Biol. 3, 523–530
3 Yang, K., Bai, H. J., Qi, O. Y., Lai, L. H. and Tang, C. (2008) Finding multiple target
optimal intervention in disease-related molecular network. Mol. Syst. Biol. 4, 228
4 Jiang, X. L., Zhou, L., Wei, D. G., Meng, H., Liu, Y. and Lai, L. H. (2008) Activation and
inhibition of leukotriene A(4) hydrolase aminopeptidase activity by diphenyl ether and
derivatives. Bioorg. Med. Chem. Lett. 18, 6549–6552
5 Jiang, X. L., Zhou, L., Wu, Y. R., Wei, D. G., Sun, C. Y., Jia, J., Liu, Y. and Lai, L. H. (2010)
Modulating the substrate specificity of LTA4H aminopeptidase by using chemical
compounds and small-molecule-guided mutagenesis. ChemBioChem 11, 1120–1128
6 Hara, S., Kamei, D., Sasaki, Y., Tanemoto, A., Nakatani, Y. and Murakami, M. (2010)
Prostaglandin E synthases: understanding their pathophysiological roles through mouse
genetic models. Biochimie 92, 651–659
7 Hammarberg, T., Hamberg, M., Wetterholm, A., Hansson, H., Samuelsson, B. and
Haeggstrom, J. Z. (2009) Mutation of a critical arginine in microsomal prostaglandin E
synthase-1 shifts the isomerase activity to a reductase activity that converts prostaglandin
H-2 into prostaglandin F-2α. J. Biol. Chem. 284, 301–305
8 Murakami, M., Naraba, H., Tanioka, T., Semmyo, N., Nakatani, Y., Kojima, F., Ikeda, T.,
Fueki, M., Ueno, A., Ohishi, S. et al. (2000) Regulation of prostaglandin E-2 biosynthesis
by inducible membrane-associated prostaglandin E-2 synthase that acts in concert with
cyclooxygenase-2. J. Biol. Chem. 275, 32783–32792
9 Pawelzik, S. C., Uda, N. R., Spahiu, L., Jegerschold, C., Stenberg, P., Hebert, H.,
Morgenstern, R. and Jakobsson, P. J. (2010) Identification of key residues determining
species differences in inhibitor binding of microsomal prostaglandin E synthase-1.
J. Biol. Chem. 285, 29254–29261
10 Jakobsson, P. J., Morgenstern, R., Mancini, J., Ford-Hutchinson, A. and Persson, B.
(2000) Membrane-associated proteins in eicosanoid and glutathione metabolism
(MAPEG): a widespread protein superfamily. Am. J. Resp. Crit. Care 161, S20–S24
11 Holm, P. J., Morgenstern, R. and Hebert, H. (2002) The 3-D structure of microsomal
glutathione transferase 1 at 6 angstrom resolution as determined by electron
crystallography of p22(1)2(1) crystals. Biochim. Biophys Acta 1594, 276–285
12 Holm, P. J., Bhakat, P., Jegerschold, C., Gyobu, N., Mitsuoka, K., Fujiyoshi, Y.,
Morgenstern, R. and Hebert, H. (2006) Structural basis for detoxification and oxidative
stress protection in membranes. J. Mol. Biol. 360, 934–945
13 Hamza, A., Abdulhameed, M.D.M. and Zhan, C. G. (2008) Understanding microscopic
binding of human microsomal prostaglandin E synthase-1 with substrates and inhibitors
by molecular modeling and dynamics simulation. J. Phys. Chem. B 112,
7320–7329
14 Xing, L., Kurumbail, R., Frazier, R., Davies, M., Fujiwara, H., Weinberg, R., Gierse, J.,
Caspers, N., Carter, J., McDonald, J. et al. (2009) Homo-timeric structural model of
human microsomal prostaglandin E synthase-1 and characterization of its
substrate/inhibitor binding interactions. J. Compu.-Aided Mol. Des. 23, 13–24
15 Huang, X. Q., Yan, W. L., Gao, D. Q., Tong, M., Tai, H. H. and Zhan, C. G. (2006)
Structural and functional characterization of human microsomal prostaglandin E
synthase-1 by computational modeling and site-directed mutagenesis. Bioorg. Med.
Chem. 14, 3553–3562
16 Hamza, A., Tong, M., Abdulhameed, M.D.M., Liu, J. J., Goren, A. C., Tai, H. H. and Zhan,
C. G. (2010) Understanding microscopic binding of human microsomal prostaglandin E
synthase-1 (mPGES-1) trimer with substrate PGH2 and cofactor GSH: insights from
computational alanine scanning and site-directed mutagenesis. J. Phys. Chem. B 114,
5605–5616
21
17 Ferguson, A. D., McKeever, B. M., Xu, S. H., Wisniewski, D., Miller, D. K., Yamin, T. T.,
Spencer, R. H., Chu, L., Ujjainwalla, F., Cunningham, B. R. et al. (2007) Crystal structure
of inhibitor-bound human 5-lipoxygenase-activating protein. Science 317, 510–512
18 Molina, D. M., Wetterholm, A., Kohl, A., McCarthy, A. A., Niegowski, D., Ohlson, E.,
Hammarberg, T., Eshaghi, S., Haeggstrom, J. and Nordlund, P. R. (2007) Structural basis
for synthesis of inflammatory mediators by human leukotriene C-4 synthase. Nature 448,
613–616
19 Jegerschöld, C., Paweizik, S. C., Purhonen, P., Bhakat, P., Gheorghe, K. R., Gyobu, N.,
Mitsuoka, K., Morgenstern, R., Jakobsson, P. J. and Hebert, H. (2008) Structural basis for
induced formation of the inflammatory mediator prostaglandin E-2. Proc. Natl. Acad. Sci.
U.S.A. 105, 11110–11115
20 Nordlund, P., Molina, D. M. and Eshaghi, S. (2008) Catalysis within the lipid bilayer:
structure and mechanism of the MAPEG family of integral membrane proteins. Curr. Opin.
Struct. Biol. 18, 442–449
21 Kim, W. I., Choi, K. A., Do, H. S. and Yu, Y. G. (2008) Expression and purification of
human mPGES-1 in E. coli and identification of inhibitory compounds from a
drug-library. BMB Rep. 41, 808–813
22 Thoren, S., Weinander, R., Saha, S., Jegerschold, C., Pettersson, P. L., Samuelsson, B.,
Hebert, H., Hamberg, M., Morgenstern, R. and Jakobsson, P. J. (2003) Human
microsomal prostaglandin E synthase-1: purification, functional characterization, and
projection structure determination. J. Biol. Chem. 278, 22199–22209
23 Saxl, R. L., Changchien, L. M., Hardy, L. W. and Maley, F. (2001) Parameters affecting the
restoration of activity to inactive mutants of thymidylate synthase via subunit exchange:
further evidence that thymidylate synthase is a half-of-the-sites activity enzyme.
Biochemistry 40, 5275–5282
24 Reference deleted
25 Hess, B., Kutzner, C., van der Spoel, D. and Lindahl, E. (2008) GROMACS 4: algorithms
for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory
Comput. 4, 435–447
26 Parrinello, M. and Rahman, A. (1980) Crystal-structure and pair potentials: a
molecular-dynamics study. Phys. Rev. Lett. 45, 1196–1199
27 Nose, S. (1984) A molecular-dynamics method for simulations in the canonical
ensemble. Mol. Phys. 52, 255–268
28 Darden, T., York, D. and Pedersen, L. (1993) Particle mesh Ewald: an N.Log(N) method
for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092
29 Hess, B., Bekker, H., Berendsen, H.J.C. and Fraaije, J.G.E.M. (1997) LINCS: a linear
constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472
30 Alander, J., Lengqvist, J., Holm, P. J., Svensson, R., Gerbaux, P., van den Heuvel, R.H.H.,
Hebert, H., Griffiths, W. J., Armstrong, R. N. and Morgenstern, R. (2009) Microsomal
glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione.
Arch. Biochem. Biophys. 487, 42–48
31 Nury, H., Poitevin, F., Van Renterghem, C., Changeux, J. P., Corringer, P. J., Delarue, M.
and Baaden, M. (2010) One-microsecond molecular dynamics simulation of channel
gating in a nicotinic receptor homologue. Proc. Natl. Acad. Sci. U.S.A. 107,
6275–6280
32 Marianayagam, N. J., Sunde, M. and Matthews, J. M. (2004) The power of two: protein
dimerization in biology. Trends Biochem. Sci. 29, 618–625
33 Renatus, M., Stennicke, H. R., Scott, F. L., Liddington, R. C. and Salvesen, G. S. (2001)
Dimer formation drives the activation of the cell death protease caspase 9. Proc. Natl.
Acad. Sci. U.S.A. 98, 14250–14255
34 Terrillon, S. and Bouvier, M. (2004) Roles of G-protein-coupled receptor dimerization:
from ontogeny to signalling regulation. EMBO Rep. 5, 30–34
35 Lai, L. H., Li, C. M., Qi, Y. F., Teng, X., Yang, Z. C., Wei, P., Zhang, C. S., Tan, L., Zhou, L.
and Liu, Y. (2010) Maturation mechanism of severe acute respiratory syndrome (SARS)
coronavirus 3C-like proteinase. J. Biol. Chem. 285, 28134–28140
36 Noji, H., Yasuda, R., Yoshida, M. and Kinosita, K. (1997) Direct observation of the rotation
of F-1-ATPase. Nature 386, 299–302
37 Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K. and Schramm, V. L. (1998)
One-third-the-sites transition-state inhibitors for purine nucleoside phosphorylase.
Biochemistry 37, 8615–8621
38 Griffin, M.D.W., Dobson, R.C.J., Pearce, F. G., Antonio, L., Whitten, A. E., Liew, K.,
Mackay, J. P., Trewhella, J., Jameson, G. B., Perugini, M. A. et al. (2008) Evolution of
quaternary structure in a homotetrameric enzyme. J. Mol. Biol. 380, 691–703
39 Popot, J. L. and Engelman, D. M. (1990) Membrane-protein folding and oligomerization:
the 2-stage model. Biochemistry 29, 4031–4037
40 Yuan, Y., Pei, J. and Lai, L. (2011) LigBuilder 2: a practical de novo drug design
approach. J. Chem. Inf. Model. 51, 1083–1091
Received 1 June 2011/29 July 2011; accepted 29 July 2011
Published as BJ Immediate Publication 29 July 2011, doi:10.1042/BJ20110977
c The Authors Journal compilation c 2011 Biochemical Society
Biochem. J. (2011) 440, 13–21 (Printed in Great Britain)
doi:10.1042/BJ20110977
SUPPLEMENTARY ONLINE DATA
Microsomal prostaglandin E synthase-1 exhibits one-third-of-the-sites
reactivity
Shan HE*, Yiran WU*, Daqi YU* and Luhua LAI*†1
*Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering,
Peking University, Beijing 100871, China, and †Center for Theoretical Biology, AAIS, Peking University, Beijing 100871, China
Figure S1
Construction of the open conformation structural model of mPGES-1
(A) Flowchart of model construction (see Supplementary Table S1 for detailed MD simulation steps). (B) MD simulation box containing three protein chains (magenta), three GSH moieties (yellow),
98 POPC moieties (green), 3877 water molecules (red) and 21 Cl − ions (blue). (C) RMSD of monomers, GSH and PGH2 with respect to the starting pose during the last 20 ns of the fully relaxed MD
simulation. The RMSD of PGH2 was the highest, corresponding with its large mobility. All of the RMSD values were below 3 Å, indicating that the MD simulation was stable. (D) The Ramachandran
plot of our mPGES-1 model. The G-factor grew from − 0.29 (PDB code 3DWW) to − 0.10, indicating a more reasonable structure.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
S. He and others
Figure S2
Comparison of our mutation result to published mutation data
(A) Unimportant residues (defined as residues that after their mutation, mPGES-1 activity remained above 50 %) identified by published mutation data [17–20]. In pocket-2 of our model, the
unimportant residues were not located near the substrate-binding pocket, in good agreement with the experimental results. (B) Comparison of our mutation result to published data [17,19,21]. R38A
and R38S have not been used previously. The important residues identified in our model were also supported by published mutagenesis data, excluding Y117F (full activity in [17]). However, as it
could form hydrogen bonds with GSH and was in a radius of 5 Å around the endoperoxide bridge of PGH2 , its mutation should have a great effect on the enzymatic activity. Our mutagenesis produced
a 70 % decrease in activity, supporting its importance.
Figure S3
Quantification of mPGES-1 content in the microsome samples and co-IP control experiments
(A) mPGES1 content in the microsome samples of mutants and WT were analysed by anti-mPGES-1 Western blotting (WB). The Western blot membrane was imaged using the Bio-Rad Laboratories
imaging instrument (ChemiDoc EQ), and the band density was quantified using the software package Quantity One® 4.6.2. Density and volume contour tools were used to identify and quantify
bands. The top panel illustrates what each band represented, and the red arrow indicates the position of the mPGES-1 band at approximately 16 kDa. The bottom panel shows the density tool analysis
result in which signal intensity distributions were displayed along the top and side of the image. The Quantity One® software quantified protein bands using the following formula: quantity = sum of
the intensity of a pixel × pixel size for all pixels in the boundary. Therefore the resulting values have units of intensity × mm2 . The analysis results for each band are listed in Supplementary Table
S3. The results are average values of three parallel repeats, and only one set of blots is shown in the Figure. (B) Enzymatic activity assay of the negative control and different species of mPGES-1
(R126A, His–R126A, WT and His–WT) that were used in the co-IP. The x axis represented the total protein concentration of the microsome samples determined by the bicinchoninic acid protein
assay. As WT and His–WT had comparable activity, the attached His6 tag should have no effect on the enzymatic activity. The mutants R126A and His–R126A had no detectable activity, similar to the
negative control. Results are means +
− S.E.M. (n = 3). (C) Anti-mPGES-1 Western blot analysis with the molecular mass marker (lane 1), negative control (NC; lane 2) and positive control (lane 3;
PC, mPGES-1 Western-ready control purchased from Cayman Chemical). In lanes 3–5, positive control, WT and R126A microsome sample bands had similar molecular mass (theoretical molecular
mass of 16 kDa). In lanes 6–7, His–WT and His–R126A microsome sample bands had similar molecular masses (theoretical molecular mass of 18 kDa). Lanes 8–10 are samples containing equal
amounts of WT and R126A mixed for 28 h and then immunoprecipitated with anti-mPGES-1 antibody (mPGES IP*) or anti-His6 -tag antibody (His IP). Both the immunoprecipitated proteins (IP)
and the supernatant (S) were analysed. The mPGES IP could represent the total amount of mPGES-1 (WT and R126A) in the samples. In contrast, the His IP contained no detectable mPGES-1, with
almost all mPGES-1 remaining in the supernatants. This result indicated that there was no cross-reaction between mPGES-1 (WT or R126A) and the His6 -tag antibody. Molecular mass is given in
kDa on the left-hand side.
c The Authors Journal compilation c 2011 Biochemical Society
One-third-of-the-sites reactivity of mPGES-1
Figure S4 Calculation method of the theoretical activity of the enzyme
mixture for three possible enzymatic mechanisms
(A) Calculated proportion of different species of mPGES-1 (W represents the WT monomer
and M represents the mutant R126A monomer) in the total mPGES-1 mixture at various molar
ratios, with the assumption that WT and R126A have the same capability of forming trimers
(as Arg126 was located far from the subunit interface, its mutation might not influence the
formation of the hybrid trimer, which was supported by the experimental results). The Figure
shows that with increasing M/W molar ratios, the proportion of WWW quickly declined, whereas
that of MMM increased. The proportion of hybrid trimers WWM and WMM initially increased
and then declined. (B) Calculated activity of the enzyme mixture for three possible enzymatic
mechanisms and experimental data. The activity of the first point (m = 0) of the WT + R126A
activity curve served as the control (100 % activity) and was used for comparison of the activity
at other points (relative activity). The activity of the enzyme mixture could be calculated by
adding the activities of all active species. There are three possible enzymatic mechanisms: (i)
all-sites reactivity (only WWW has activity); (ii) two-thirds-of-sites reactivity (WWW and WWM
have activity); and (ii) one-third-of-sites reactivity (WWW, WWM and WMM have activity).
When m = 1, the total protein quantity (M + W) equals 2. Then, the activity for the all-sites
mechanism would be 2 × 1/8 = 1/4, where 1/8 is the proportion of the active species WWW.
The activity for the two-thirds-of-sites mechanism would be 2 × 1/8 + 2 × 3/8 = 1, where 1/8
and 3/8 are the proportions of the active species WWW and WWM respectively. The activity
for the one-third-of-sites mechanism would be 2 × 1/8 + 2 × 3/8 + 2 × 3/8 = 7/4. (It should
be noted that as the ‘one-third-of-sites reactivity’ meant mPGES-1 could have only one pocket
active at any given time, the enzymatic activities of WWW, WWM and WMM were assumed
to be comparable. Castellani et al. [22] reported that the dimeric cytochrome bc1 exhibits
half-of-the-sites reactivity and the activity of the heterodimer was essentially identical with the
WT homodimer. Therefore the assumption was reasonable.) Other points were calculated in
the same manner. The calculated values are listed in Supplementary Table S5 and plotted
in the Figure. The Figure shows that with increasing M/W molar ratios, only the activity
under the one-third-of-sites reactivity assumption increased, whereas it decreased in the other
two situations (under the two-thirds-of-sites reactivity assumption, the activity might initially
increase and then decline in the range of M/W = 0–1. However, in the entire range, an overall
decline was shown). The experimental results WT + R126A were in good agreement with
the one-third-of-sites reactivity assumption. We also performed a control experiment using
WT + negative control, in which the activity was nearly unchanged by varying the M/W molar
ratio.
c The Authors Journal compilation c 2011 Biochemical Society
S. He and others
Figure S5
Incomplete conformation transition from closed to open
(A) After-MD structure of simulation B + (extended to 100 ns). The distance between the α-carbon atoms of Leu39 and Arg126 was used to represent the extent of opening of the substrate-binding
pocket. Pocket-2 was only open partially (the Leu39 –Arg126 distance grew from 8 Å to 12 Å), and PGH2 could not dock within the binding pocket. A molecule of PGH2 (blue) was manually placed to
illustrate the pocket entrance. Residues that sterically hindered substrate binding (Tyr28 , Ile32 , Leu39 , Tyr130 , Thr131 , Leu135 and Ala138 ) were labeled. The Tyr28 –Tyr130 pair acted as gatekeepers, and
the residues identified by Pawelzik et al. [23] as crucial residues for substrate/inhibitor binding was also located in this region. (B) The Leu39 –Arg126 distance variation during simulation B + (top
panel) and simulation C (bottom panel). In both MD simulations, no pockets transformed to the fully open conformation (Leu39 –Arg126 distance ≈ 13.4 Å).
Table S1
MD simulation steps for construction of the mPGES-1 open conformation structural model
The proportion was calculated using the formula: f (k , n , p ) = C nk p k (1 − p )n −k where, k is the amount of M in the formed trimer; n equals 3; and p is the proportion of M in the total mPGES-1
mixture. The parameter p changed with various M/W molar ratios (m). When m = 0, p = 0 and m = 10 then p = 10/11. NPT, (constant number of particles, pressure and temperature); NVT,
(constant particle number, volume and temperature).
Main steps
Optimizing steps
Optimized atoms
Integrator
Maximum force (kJ/mol)
1. Energy minimization
1-1
1-2
1-3
1-4
1-5
1-6
1-7
1-8
H
H
Loop
Loop
H2 O
POPC
Protein and GSH
All system
Steep
Cg (conjugate gradient)
Steep
Cg (conjugate gradient)
Steep
Steep
Steep
Cg (conjugate gradient)
1000
100
1000
100
1000
1000
1000
100
MD
Optimizing steps
Optimized atoms
Restraint (kJ·mol − 1 ·nm − 2 )
Time (ns)
2. NVT equilibration phase
3. NPT equilibration phase
4. Stabilizing GSH
7. Fully relaxed
2
3
4-1
4-2
4-3
4-4
4-5
5
6-1
6-2
6-3
7
All system
All system
POPC, H2 O and Cl −
Residues
Residues
GSH
GSH
PGH2
POPC, H2 O and Cl −
POPC, H2 O and Cl −
GSH
All system
1000
1000
Protein and GSH 1000
Cα and GSH 500
Cα and GSH 100
Cα 100 GSH 10
Cα 100
–
Protein, GSH and PGH2 1000
Protein, GSH and PGH2 100
PGH2 100 GSH 10
None
1
5
5
10
10
10
20
–
5
5
10
20
MD
Optimizing steps
Optimized atoms
Integrator
Maximum force (kJ/mol)
8. Energy minimization
8-1
8-2
8-3
8-4
H2 O
POPC
All system
All system
Steep
Steep
Steep
Cg (conjugate gradient)
1000
1000
1000
100
5. PGH2 dock
6. Stabilizing PGH2
c The Authors Journal compilation c 2011 Biochemical Society
One-third-of-the-sites reactivity of mPGES-1
Table S2
The 19 currently published types of mPGES-1 inhibitors used in the docking studies
Representative compound name*
Derivative
MK-886
30
27
26
23
28
25
21
24
33
13
12
10
18
19
11
1
17
16
15
8
4
6
2
3
7
MF63
16e
16a
8
16b
5
2a
2k
2i
2g
2l
13
15
11
7
2h
6
10
2c
2d
9
Oxicam
13j
13m
13k
13l
13g
31
10
13d
13c
17
33
36
34
13a
13b
30
13f
13n
32
13i
20
Included numbers
IC50 range (μM)
Reference
25
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
No. 7
No. 8
No. 9
No. 10
No. 11
No. 12
No. 13
No. 14
No. 15
No. 16
No. 17
No. 18
No. 19
No. 20
No. 21
No. 22
No. 23
No. 24
No. 25
20
No. 26
No. 27
No. 28
No. 29
No. 30
No. 31
No. 32
No. 33
No. 34
No. 35
No. 36
No. 37
No. 38
No. 39
No. 40
No. 41
No. 42
No. 43
No. 44
No. 45
21
No. 46
No. 47
No. 48
No. 49
No. 50
No. 51
No. 52
No. 53
No. 54
No. 55
No. 56
No. 57
No. 58
No. 59
No. 60
No. 61
No. 62
No. 63
No. 64
No. 65
No. 66
>0.003
0.003
0.004
0.005
0.007
0.008
0.012
0.016
0.032
0.07
0.25
0.26
0.29
0.33
0.6
0.9
1.6
2.6
3.2
4.3
6.4
6.7
7.2
>10
>10
>10
>0.001
0.009
0.051
0.073
0.075
0.087
0.14
0.33
0.41
0.53
0.56
0.7
0.71
1.3
2
2.5
6.6
8
>10
>10
>10
>0.016
0.016
0.026
0.038
0.043
0.07
0.098
0.11
0.11
0.15
0.16
0.18
0.19
0.28
0.29
0.53
0.69
0.86
1
3.05
3.13
11.7
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[1]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[2]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
[3]
c The Authors Journal compilation c 2011 Biochemical Society
S. He and others
Table S2
Continued
Representative compound name*
Derivative
Pirinixic acid
7b
7a
7d
7e
4
2d
2h
3b
8
1
2a
2c
2e
5c
7c
Benzo[g]indol-3-carboxylates
9
7c
7b
7a
7d
6
13
14
5
8
10
2
3
4
Licofelone
10c
11f
14
11c
11d
10f
3
10b
10a
AF3442
Curcumin
Arachidonic acid
Thienopyrrole
No. 18
MC
Naphthalene disulfonamide
Garcinal
Benzoxazole
EGCG
Dimethylcelecoxib
NS398
PGJ2
Included numbers
IC50 range (μM)
Reference
15
No. 67
No. 68
No. 69
No. 70
No. 71
No. 72
No. 73
No. 74
No. 75
No. 76
No. 77
No. 78
No. 79
No. 80
No. 81
14
No. 82
No. 83
No. 84
No. 85
No. 86
No. 87
No. 88
No. 89
No. 90
No. 91
No. 92
No. 93
No. 94
No. 95
9
No. 96
No. 97
No. 98
No. 99
No. 100
No. 101
No. 102
No. 103
No. 104
No. 105
No. 106
No. 107
No. 108
No. 109
No. 110
No. 111
No. 112
No. 113
No. 114
No. 115
No. 116
No. 117
>1.3
1.3
1.6
1.7
2.1
2.6
3.9
5.1
5.6
>10
>10
>10
>10
>10
>10
>10
>0.1
0.1
0.2
0.5
0.6
0.6
1.6
1.7
2.8
3.1
3.4
9.2
>10
>10
>10
>1.8
1.8
2.1
3.9
4.5
4.8
6.2
6.7
7.2
>10
0.06
0.3
0.3
0.39
0.5
1
1.1
1.2
1.3
1.8
15.6
20
55
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[4]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[6]
[6]
[6]
[6]
[6]
[6]
[6]
[6]
[6]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[10]
[13]
[10]
[14]
[15]
[16]
[9]
*The compound names were in accordance with those in the original literature. Here, all compounds were rearranged according to ascending IC50 values. Eight compounds of the MK-886 series
and eleven compounds of the MF63 series were removed to achieve uniform data distribution.
c The Authors Journal compilation c 2011 Biochemical Society
One-third-of-the-sites reactivity of mPGES-1
Table S3
Analysis of the Western blotting results
The Western blots are shown in Supplementary Figure S3(A). Results are means +
− S.E.M.
(n = 3).
mPGES-1 variant
Quantity/intensity × mm2
R38A
R38S
H72A
N74A
E77A
R110A
R126A
Y117A
Y117F
WT
2186.57 +
− 26.75
2381.93 +
− 8.20
2123.01 +
− 42.53
750.75 +
− 38.54
587.15 +
− 2.13
534.86 +
− 14.92
1037.50 +
− 12.36
2127.74 +
− 22.73
1399.84 +
− 10.85
2027.82 +
− 18.64
Table S4
Calculated proportion of different species of mPGES-1 at varying molar ratios
The proportion was calculated using the formula: f (k , n , p ) = C nk p k (1 − p )n −k , where k is the amount of M in the formed trimer; n equals 3; and p is the proportion of M in the total mPGES-1
mixture. The parameter p changed with various M/W molar rations (m). When m = 0, p = 0 and m = 10 then p = 10/11.
Table S5
R126A/WT molar ratio
WWW (k = 0)
WWM (k = 1)
WMM (k = 2)
MMM (k = 3)
0 (p = 0)
1 (p = 1/2)
2 (p = 2/3)
5 (p = 5/6)
10 (p = 10/11)
1
1/8
1/27
1/216
1/1331
0
3/8
6/27
15/216
30/1331
0
3/8
12/27
75/216
300/1331
0
1/8
8/27
125/216
1000/1331
Calculated activity of the enzyme mixture at various molar ratios for three possible enzymatic mechanisms
R126A/WT molar ratio
Enzymatic activity (all-sites)
Enzymatic activity (two-thirds-of-sites)
Enzymatic activity (one-third-of-sites)
0
1
2
5
10
1
1/4
1/9
1/36
1/121
1
1
7/9
4/9
31/121
1
7/4
19/9
91/36
331/121
REFERENCES
1 Riendeau, D., Aspiotis, R., Ethier, D., Gareau, Y., Grimm, E. L., Guay, J., Guiral, S.,
Juteau, H., Mancini, J. A., Methot, N. et al. (2005) Inhibitors of the inducible microsomal
prostaglandin E-2 synthase (mPGES-1) derived from MK-886. Bioorg. Med. Chem. Lett.
15, 3352–3355
2 Cote, B., Boulet, L., Brideau, C., Claveau, D., Ethier, D., Frenette, R., Gagnon, M.,
Giroux, A., Guay, J., Guiral, S. et al. (2007) Substituted phenanthrene imidazoles as
potent, selective, and orally active mPGES-1 inhibitors. Bioorg. Med. Chem. Lett. 17,
6816–6820
3 Wang, J., Limburg, D., Carter, J., Mbalaviele, G., Gierse, J. and Vazquez, M. (2010)
Selective inducible microsomal prostaglandin E-2 synthase-1 (mPGES-1) inhibitors
derived from an oxicam template. Bioorg. Med. Chem. Lett. 20, 1604–1609
4 Koeberle, A., Zettl, H., Greiner, C., Wurglics, M., Schubert-Zsilavecz, M. and Werz, O.
(2008) Pirinixic acid derivatives as novel dual inhibitors of microsomal prostaglandin E-2
synthase-1 and 5-lipoxygenase. J. Med. Chem. 51, 8068–8076
5 Koeberle, A., Haberl, E. M., Rossi, A., Pergola, C., Dehm, F., Northoff, H., Troschuetz, R.,
Sautebin, L. and Werz, O. (2009) Discovery of benzo[g]indol-3-carboxylates as potent
inhibitors of microsomal prostaglandin E-2 synthase-1. Bioorg. Med. Chem. 17,
7924–7932
6 Liedtke, A. J., Keck, P. R., Lehmann, F., Koeberle, A., Werz, O. and Laufer, S. A. (2009)
Arylpyrrolizines as inhibitors of microsomal prostaglandin E-2 synthase-1 (mPGES-1) or
as dual inhibitors of mPGES-1 and 5-lipoxygenase (5-LOX). J. Med. Chem. 52,
4968–4972
7 Bruno, A., Di Francesco, L., Coletta, I., Mangano, G., Alisi, M. A., Polenzani, L., Milanese,
C., Anzellotti, P., Ricciotti, E., Dovizio, M. et al. (2010) Effects of AF3442
[N -(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human
microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human
monocytes in vitro . Biochem. Pharmacol. 79, 974–981
8 Koeberle, A., Northoff, H. and Werz, O. (2009) Curcumin blocks prostaglandin E-2
biosynthesis through direct inhibition of the microsomal prostaglandin E-2 synthase-1.
Mol. Cancer Ther. 8, 2348–2355
9 Quraishi, O., Mancini, J. A. and Riendeau, D. (2002) Inhibition of inducible prostaglandin
E-2 synthase by 15-deoxy-δ(12,14)-prostaglandin J(2) and polyunsaturated fatty acids.
Biochem. Pharmacol. 63, 1183–1189
10 Friesen, R. W. and Mancini, J. A. (2008) Microsomal prostaglandin E-2 synthase-1
(mPGES-1): a novel anti-inflammatory therapeutic target. J. Med. Chem. 51, 4059–4067
11 Rorsch, F., Wobst, I., Zettl, H., Schubert-Zsilavecz, M., Grosch, S., Geisslinger, G.,
Schneider, G. and Proschak, E. (2010) Nonacidic inhibitors of human microsomal
prostaglandin synthase 1 (mPGES 1) identified by a multistep virtual screening protocol.
J. Med. Chem. 53, 911–915
12 Koeberle, A., Pollastro, F., Northoff, H. and Werz, O. (2009) Myrtucommulone, a natural
acylphloroglucinol, inhibits microsomal prostaglandin E-2 synthase-1. Br. J. Pharmacol.
156, 952–961
13 Koeberle, A., Northoff, H. and Werz, O. (2009) Identification of 5-lipoxygenase and
microsomal prostaglandin E-2 synthase-1 as functional targets of the anti-inflammatory
and anti-carcinogenic garcinol. Biochem. Pharmacol. 77, 1513–1521
14 Koeberle, A., Bauer, J., Verhoff, M., Hoffmann, M., Northoff, H. and Werz, O. (2009) Green
tea epigallocatechin-3-gallate inhibits microsomal prostaglandin E-2 synthase-1.
Biochem. Biophys. Res. Commun. 388, 350–354
c The Authors Journal compilation c 2011 Biochemical Society
S. He and others
15 Wobst, I., Schiffmann, S., Birod, K., Maier, T. J., Schmidt, R., Angioni, C., Geisslinger, G.
and Grosch, S. (2008) Dimethylcelecoxib inhibits prostaglandin E-2 production.
Biochem. Pharmacol. 76, 62–69
16 Thoren, S. and Jakobsson, P. J. (2000) Coordinate up- and down-regulation of
glutathione-dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells:
inhibition by NS-398 and leukotriene C-4. Eur. J. Biochem. 267, 6428–6434
17 Jegerschold, C., Paweizik, S. C., Purhonen, P., Bhakat, P., Gheorghe, K. R., Gyobu, N.,
Mitsuoka, K., Morgenstern, R., Jakobsson, P. J. and Hebert, H. (2008) Structural basis for
induced formation of the inflammatory mediator prostaglandin E-2. Proc. Natl. Acad. Sci.
U.S.A. 105, 11110–11115
18 Huang, X. Q., Yan, W. L., Gao, D. Q., Tong, M., Tai, H. H. and Zhan, C. G. (2006)
Structural and functional characterization of human microsomal prostaglandin E
synthase-1 by computational modeling and site-directed mutagenesis. Bioorg. Med.
Chem. 14, 3553–3562
19 Hammarberg, T., Hamberg, M., Wetterholm, A., Hansson, H., Samuelsson, B. and
Haeggstrom, J. Z. (2009) Mutation of a critical arginine in microsomal prostaglandin
E synthase-1 shifts the isomerase activity to a reductase activity that converts
prostaglandin H-2 into prostaglandin F-2α. J. Biol. Chem. 284, 301–305
Received 1 June 2011/29 July 2011; accepted 29 July 2011
Published as BJ Immediate Publication 29 July 2011, doi:10.1042/BJ20110977
c The Authors Journal compilation c 2011 Biochemical Society
20 Murakami, M., Naraba, H., Tanioka, T., Semmyo, N., Nakatani, Y., Kojima, F., Ikeda, T.,
Fueki, M., Ueno, A., Ohishi, S. et al. (2000) Regulation of prostaglandin E-2
biosynthesis by inducible membrane-associated prostaglandin E-2 synthase
that acts in concert with cyclooxygenase-2. J. Biol. Chem. 275, 32783–32792
21 Hamza, A., Tong, M., Abdulhameed, M. D. M., Liu, J. J., Goren, A. C., Tai, H. H. and
Zhan, C. G. (2010) Understanding microscopic binding of human microsomal
prostaglandin E synthase-1 (mPGES-1) trimer with substrate PGH2 and cofactor GSH:
insights from computational alanine scanning and site-directed mutagenesis.
J. Phys. Chem. B 114, 5605–5616
22 Castellani, M., Covian, R., Kleinschroth, T., Anderka, O., Ludwig, B. and Trumpower, B. L.
(2010) Direct demonstration of half-of-the-sites reactivity in the dimeric cytochrome bc1
complex enzyme with one inactive monomer is fully active but unable to activate the
second ubiquinol oxidation site in response to ligand binding at the ubiquuinone
reduction site. J. Biol. Chem. 285, 502–510
23 Pawelzik, S. C., Uda, N. R., Spahiu, L., Jegerschold, C., Stenberg, P., Hebert, H.,
Morgenstern, R. and Jakobsson, P. J. (2010) Identification of key residues determining
species differences in inhibitor binding of microsomal prostaglandin E synthase-1.
J. Biol. Chem. 285, 29254–29261