Identification of residues in the PXR ligand binding domain critical
for species specific and constitutive activation
Tove O
¨
stberg
1,
*, Go¨ ran Bertilsson
1,
*
,†
, Lena Jendeberg
2
, Anders Berkenstam
2,‡
and Jonas Uppenberg
3
1
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden;
2
Departments of Biology, and
3
Structural Chemistry, Biovitrum, Stockholm, Sweden
The cytochrome P450 family of enzymes has long been
known to metabolize a wide range of compounds, including
many of today’s most common drugs. A novel nuclear
receptor called PXR has been established as an activator of
several of the cytochrome P450 genes, including CYP3A4.
This enzyme is believed to account for the metabolism of
more than 50% of all prescription drugs. PXR is therefore
used as a negative selector target and discriminatory filter in
preclinical drug development.

animals not only become more sensitive to xenobiotics but
also fail to induce CYP3A by known PXR activators [6].
PXR heterodimerizes with 9-cis-retinoic acid receptors
(RXR, NR1B1-3) and binds and induces gene expression
through a specific genomic response element in the promo-
ter region of CYP3A4 and CYP3A7 [1–3,7–9]. PXR is
closely related to the constitutive androstane receptor
(CAR, NR1I3), which is believed to have a complementary
role to PXR in the genetic regulation of cytochrome P450
expression. CAR has been established as a CYP2B gene
regulator [10–12], but also activates the same genomic
response elements in CYP3A4 and CYP3A7 as PXR [9,13].
PXR has been shown to bind phenobarbital response
elements in the CYP2B gene promoter and to be a regulator
of CYP2B10 [14] and CYP2B6 gene transcription [15]. The
PXR receptor exhibit a promiscuous ligand dependent
activation profile and a broad range of synthetic xenobiotics
are known to activate the receptor [1–4]. In addition to the
activation of PXR by exogenous xenobiotics, it was recently
shown that also the endogenously produced, but highly
hepatotoxic cholesterol derivative litocholic acid is a potent
activator of PXR [16,17]. Accordingly, PXR is involved not
only in the detoxification of exogenous xenobiotics, but also
of endogenously produced substances.
Cloning of PXR orthologs from human, rabbit, rat and
mouse [18] has shown that the ligand-binding domain has
diverged considerably between the different species. The
species divergence and specific activation profile of the
orthologous PXRs have also been shown to reflect species
specific differences in CYP3A gene induction. For example,

the potential to predict species differences in response to
xenobiotics [6]. Structural insights into the molecular
mechanism of PXR activation will increase the understand-
ing of these species differences and may be used in structure
based drug design to avoid PXR activation with its
potentially linked side-effects, such as drug-interactions,
drug-induced hepatomegaly and decreased bile acid excre-
tion [16].
The aim of this study was to explore the molecular
mechanism of ligand binding and activation of PXR by
modeling and site-directed mutation of the PXR ligand
binding domain (LBD). In particular, we wanted to identify
residues responsible for the observed differences between
rodents and man in order to construct human PXR mutants
with mouse like properties and vice versa. In this study we
focused on identifying polar amino acids involved in ligand
binding. Transient transfection in combination with site
directed mutagenesis of the PXR LBDs have enabled us to
identify one amino-acid residue involved in the species
specific response to activators. An intriguing and more
unexpected result was the identification of an amino-acid
position in the PXR structure that dramatically affects the
basal activity of both the human and mouse receptors.
MATERIALS AND METHODS
Plasmid constructs, human PXR
The full length cloning of human nuclear receptor
hPXR (hPAR-2) and the expression vector construct
(pcDNA3, Invitrogen) of hPXR have been described
previously [2]. Mutants of the human nuclear receptor
hPXR (PAR-2) were obtained by Transformer Site-directed

plasmid for transfection normalization was previously
described [2]. All constructs were verified by sequence
analysis.
Reporter gene assay
All transient transfection experiments were performed in
C3A cells (ATCC, CRL-10741, lot
i
1414101) in 6-well plates.
C3A cells were seeded at a concentration of 5 · 10
5
cells in
each well and incubated for 24 h at 37 °Cin2mLgrowth
medium containing minimal essential medium (MEM),
10% fetal bovine serum, nonessential amino acids and
sodium pyruvate (Life Technologies). The medium was
replaced with 2 mL transfection medium (MEM, 10%
charcoal/dextran treated fetal bovine serum (Hyclone),
nonessential amino acids, sodium pyruvate) and the cells
were cotransfected with 2 lg CYP3A4-luciferase reporter,
0.05 lg hPXR/mPXR/mutant plasmid and 0.1 lgRSV-AF
plasmid (alkaline phosphatase activity was used for nor-
malization of transfection efficiency) using FuGENE-6
(Roche) according to the manufacturer’s instructions. After
20–24 h, medium was replaced and cells were induced with
rifampicin (Sigma), SR12813 (synthesized by Biovitrum) or
Pregnenolone-16a-carbonitrile (PCN) (Sigma) in optimized
serial dilutions as indicated in the figures. DMSO was used
as vehicle. Following 48 h incubation, the medium was
analyzed for alkaline phosphatase activity according to the
manufacturer’s recommendations (Great EscAPe SEAP,

,10m
M
KCl, 0.5 m
M
dithiothreitol, 1 m
M
EDTA, 1 m
M
EGTA, 1% Triton X-100, protease inhibi-
tors). Nuclear pellets were collected by centrifugation at
4000 r.p.m. for 10 min (4 °C). The supernatants were
cleared by centrifugation at 140 00 r.p.m. for 10 min
(4 °C) and saved as cytoplasmic fractions. The nuclear
pellets were resuspended in Lysis buffer B (20 m
M
Hepes/
KOH pH 7.6, 1.5 m
M
MgCl
2
,420 m
M
NaCl, 1 m
M
EDTA,
Ó FEBS 2002 Mutagenesis of PXR ligand binding domain (Eur. J. Biochem. 269) 4897
1m
M
EGTA, 20% glycerol and protease inhibitors) and
gently mixed for 20 min at 4 °C. The insoluble fractions

(Santa Cruz), diluted 1/100 in NaCl/P
i
/Tween supplemented
with 5% dry milk. The membrane was washed with NaCl/
P
i
/Tween and subsequently incubated for 45 min with
Peroxidase-conjugated rabbit anti-(goat IgG) Ig (DAKO),
diluted 1/2000 in NaCl/P
i
/Tween supplemented with 5%
dry milk. A final wash was made with NaCl/P
i
/Tween. All
NaCl/P
i
/Tween used in Western blot analysis detected by
the mPXR specific antibody was supplemented with both
5% dry milk and 5% fetal bovine serum. The Western
blot was visualized using ECL Western blotting detection
reagent RPN 2106 (Amersham Pharmacia Biotech) and
Hyperfilm ECL (Amersham Pharmacia Biotech).
Modeling
The structure of the vitamin D receptor ligand binding
domain [19] was used as template for modeling human PXR
(PDB entry 1DB1). Modeling was performed with the
program
O
[20]. The conserved residues in VDR and PXR
were kept intact in the PXR model. Substituted amino acids

with an approximate size of 15 · 5 · 5A
˚
. The binding
pocket as found by the program Voidoo was delimited by
atoms from the following residues: Leu240, Met243,
Ala244, Met246, Ser247, Phe251, Phe281, Cys284,
Gln285, Phe288, Trp299, Tyr306, Thr311, Gly314,
Phe315, Leu319, Met323, His407, Leu411, Ile414, Gln415,
Ile417, His418, Phe420, Ala421, Met425, Gln426 and
Phe429. Of these amino-acid residues we identified two
polar residues, Gln285 and His407, which were not
conserved between the mouse and human receptors and
where the side chains lined the ligand binding pocket
(Fig. 1). We proceeded to construct mutants of these two
residues based on the hypothesis that they were involved in
the species specific activator response. Three single point
mutations were made for human PXR: Q285I, H407Q and
H407A. The first two replaced the human amino-acid
residue with its mouse counterpart. The third mutant was
made in order to create a more pronounced change than the
spatially and electrostatically moderate change of a histidine
to a glutamine and thereby give additional information into
its potential role in ligand binding. We also made the three
analogous mutants of mouse PXR: I282Q, Q404H and
Q404A.
The wild-type and mutant receptors were tested in a
transient cotransfection assay, using expression vectors for
Fig. 1. The ligand binding pocket of human PXR LBD (coordinates
from the crystal structure [23] with PDB code: 1ILH). A cavity surface
was generated with the program

O
[20]. A few regions of the model
were not properly aligned due to large differences. Most of
these nonaligned regions were located in the omega-loop
and beta-sheet of the protein and contained the following
residues: 175–236, 302 and 308–320. Two short additional
loop regions were poorly modeled: residues 385–387
between helices 9 and 10 and residues 416–421 between
helices 10 and 12.
Wild-type human and mouse PXR
PCN was a strong activator of mouse PXR, while it was a
poor activator of human PXR (Fig. 3). Rifampicin and
SR12813 on the other hand showed strong activation of the
human receptor, while only weak activation of the mouse
PXR could be detected (Fig. 3).
Human Q285I and mouse I282Q
The basal reporter gene activities (i.e. in the absence of
activator) of the human Q285I and mouse I282Q receptor
Fig. 2. The structures of ligands tested for PXR activation: (A) rif-
ampicin, (B) SR12813, (C) pregnenolone-16a-carbonitrile (PCN).
Fig. 3. Diagrams of transciptional activation, as determined by luciferase
reporter assay, at two ligand concentrations for (A) human and (B)
mouse wild-type PXR. Ligand concentrations chosen were 5, 10 and/or
20 l
M
. The values have been corrected for alkaline phosphatase
activity and normalized against a DMSO control. The human receptor
was strongly activated by rifampicin (RIF) and SR12813 (SR), while
mouse PXR was primarily activated by PCN.
Ó FEBS 2002 Mutagenesis of PXR ligand binding domain (Eur. J. Biochem. 269) 4899

type (Fig. 5E). Neither rifampicin nor SR12813 activated
Q404H (data not shown).
Human H407A and mouse Q404A
The human mutant receptor H407A showed nearly a four-
fold increase in basal activity compared to wild-type and the
other mutants of the human receptor (Fig. 4A). This was
not observed for the corresponding mouse receptor mutant
Q404A, where basal activity was similar to the wild-type
(Fig. 4B). Although H407A displayed a high basal activity
it could still be activated further by rifampicin (Fig. 5B).
Also SR12813 could activate this mutant although to a
lesser extent than rifampicin. PCN however, had no effect
on this mutant (data not shown). The mouse receptor
Q404A resembled the wild-type receptor in its activation
by PCN (Fig. 5e), while showing no activation by rifampicin
or SR12813 (data not shown).
Western blots
To compare the expression levels of wild-type hPXR/
mPXR, mutant hPXR/mPXR and the endogenous expres-
sion of hPXR in C3A cells, Western blot analysis was
performed on the nuclear fractions of the cell lysates. In cells
transfected with wild-type or mutant hPXR, two bands of
similar strength were detected (Fig. 6). The band corres-
ponding to the larger protein product (approximately
54 kDa) agrees in size with the PXR isoform hPAR-2 [2].
The second band (approximately 50 kDa) corresponds in
size to hPXR-1 [1]. The amount of overexpressed protein
was similar for all four constructs. In the untransfected cells
andcellstransfectedwithemptyvector,pcDNA3(Fig. 6),a
single weak band was observed corresponding in size to

mouse PXR mutants I282Q and Q404A displayed basal activites that
were close to that of the wild-type receptor, while Q404A showed a
distinctly lower level of activation.
4900 T. O
¨
stberg et al. (Eur. J. Biochem. 269) Ó FEBS 2002
neighboring the beta sheet and what is usually referred to as
the omega loop. This could not be accurately modeled, as
the corresponding region of the template structure was not
present. As a consequence the full extent of the ligand
binding pocket was not fully modeled. We will therefore
refer to the crystal structure rather than our model in the
molecular interpretation of our results.
Western blot analysis of protein expression levels
A Western blot analysis of cell lysates containing the
human PXR constructs shows the presence of a protein of
expected size, approximately 54 kDa. However, another
band of equal strength also appears for all constructs. This
band corresponds to a protein of lower molecular mass,
approximately 50 kDa, which is comparable to a band seen
in the empty plasmid and untransfected cell control
experiments. The bands seen in the control experiments
are considerably weaker however. Some endogenous
human PXR is likely to be present in all experiments and
should be taken into account in the interpretation of the
results. However we believe that the background activity
that stems from endogenous hPXR-1 is low in comparison
with that from the transfected constructs. The second band
seen in the lanes of the transfected constructs are much
stronger than in the control experiment, suggesting instead

been improved compared to human wild-type PXR, while
the reverse mutant I282Q of the mouse receptor shows a
decreased activation by PCN. This suggests that PCN also
binds in close proximity to this residue and that a
hydrophobic interaction may be more favorable. Given
the fact that PCN is a better activator of mouse than human
PXR, we believe that this mutation is central to making the
human receptor more like the mouse receptor. This is
supported by the fact that this is the only clear example
where a hydrophilic side chain has been replaced by a
hydrophobic one in the core of the ligand binding pocket.
The Q285I mutant also shows decreased propensity for
activation by rifampicin, which indicates that this large
molecule may also come in contact with this residue. The
binding mode of rifampicin however, is unclear as it is too
large to be accommodated into the binding pocket described
by the crystal structure. The reverse mutation I282Q does
not impose enough human like properties to the mouse
receptor to make it susceptible to activation by either
rifampicin or SR12813. This suggests that while some
species specific properties may be changed by single point
mutations, others are more subtle and requires multiple
substitutions to mimic.
Mutation of human His407 and mouse Gln404
Our model suggested that this residue was located at one
end of an elongated ligand binding pocket. The crystal
structure confirmed its accessibility to ligands and the
histidine residue makes a hydrogen bond to SR12813 in one
of its binding modes. The mutation of this residue gave a
number of surprising results suggesting that this residue play

could have an influence on coactivator binding. Helix 11 is
also part of the dimerization interface and one cannot
exclude an impact on the conformation of the heterodimer
that PXR forms with RXR. It is surprising that the
analogous mutation in mouse PXR, Q404A, does not affect
basal activity, while Q404H shows a dramatic decrease of
the same. The only correlation seems to be that a histidine in
this position has a negative relative effect on basal activity.
While the effect on basal activity is striking for mutations in
this position, the ligand dependent activation is less
dramatically affected and there is little evidence to show
that this residue is important for species specific activation.
H407Q is still strongly activated by rifampicin and
SR12813, while Q404H is strongly activated by PCN. The
Fig. 6. Western blot analysis of nuclear fractions showing hPXR
expression in cells transfected with empty vector (lane 1), hPXR wild-
type (lane 2), Q285I (lane 3), H407Q (lane 4), H407A (lane 5). The
amino-acid analyses determined the protein contents loaded on the gel
as follows: empty vector (lane 1) 31 lg, hPXR wild-type (lane 2) 26 lg,
Q285I (lane 3) 34 lg, H407Q (lane 4) 35 lg, H407A (lane 5) 25 lg.
Aweakbanddetectedinthecontrolexperiment(lane1)couldbe
attributed to endogenous expression of hPXR-1. The overexpression
of hPXR-2 wild-type and mutant proteins (lanes 2–5) resulted in two
strong bands with little difference observed between the four con-
structs. The largest band corresponds to the molecular mass of hPXR-
2 (approximately 54 kDa), while the second band agrees with the
molecular mass of hPXR-1 (approximately 50 kDa). The appearance
of two gene products is most likely due to alternative translational
initiation by a non-AUG codon [33], one of which is present in the
PXR sequence [2].

helix 12 through hydrogen bonds [19,26–29]. The discovery
that His407/Gln404 plays a crucial structural role in the
activation process of PXR, could be applicable also to other
receptors and further mutational and structural studies
would be of great interest to further elucidate the dynamics
of this part of the ligand binding domain. There are other
examples where a single mutation has yielded constitutively
active nuclear receptors, such as RXR [30] and the estrogen
receptor [31,32]. In the case of RXR a mutation of Phe318
into an alanine in helix 5 causes a destabilization in a
network of hydrophobic interactions in the apo-receptor
core. In the estrogen receptor Tyr571 was mutated to an
aspartic acid in the vicinity of helix 12. This produced a
constitutively active receptor, which interacted with some
but not all coactivator proteins tested. With more structural
data on mutated nuclear receptors we may anticipate a
more detailed dynamic picture of the transition from a silent
to an activated nuclear receptor and the role of heterodimer
formation and coactivators in the relay of the transcrip-
tional signal.
ACKNOWLEDGEMENTS
We would like to thank Kristina Zachrisson for performing the amino-
acid analysis and Sven-A
˚
ke Franze
´
n, Andrea Varadi and Marianne
Israelsson for DNA sequence analysis.
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