Báo cáo Y học: Identification of residues in the PXR ligand binding domain critical for species specific and constitutive activation docx

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.

In this paper we describe the design, construction and
characterization by transient transfection of mutant recep-
tors of the human and mouse PXR ligand binding domains.
By modeling the human PXR ligand binding domain we
have identified and mutated two polar residues in the puta-
tive ligand binding pocket which differ between the human
and the mouse receptor. The first residue (Q285 in human/
I282 in mouse) was mutated between the two species with the
corresponding amino acids. These mutants showed that this
residue is important for the species specific activation ofPXR
by the ligand pregnenolone-16a-carbonitrile (PCN), while
having a less pronounced role in receptor activation by rif-
ampicin. The second residue to be mutated (H407 in human/
Q404 in mouse) unexpectedly proved to be important for the
basal level of activation of PXR. The H407A mutant of the
human receptor showed a high level of constitutive activity,
while the Q404H mutant of the mouse receptor demonstra-
ted a sharply decreased basal activity compared to wild-type.
Keywords: PXR, NR1I2, VDR, ligand binding domain,
mutagenesis.
The nuclear receptor PXR (NR1I2, PAR, SXR) has been
demonstrated to be a key determinant for the transcrip-
tional regulation of the drug metabolizing enzyme family of
heme-containing monooxygenases P450 CYP3A [1–4].
Consequently this nuclear receptor is likely to play a role
in the molecular mechanisms behind common drug inter-
actions. PXR is coexpressed in tissues where CYP3A is
induced and expressed [5]. The key role of PXRs in CYP3A
induction has been further corroborated by targeted
disruption of the mouse PXR [6]. These genetically modified

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 antibiotic compound rifampicin induces human and
rabbit, but not rodent CYP3A. It is also a ligand and
activator of the human and rabbit PXR but not the rodent
PXRs. Pregnenolone-16a-carbonitrile (PCN) on the other
hand induces rodent but not human CYP3A and likewise is
a ligand for rodent but not human PXR [13]. To date the
most potent endogenously produced PXR activator is
5b-pregnane-3,20-dione [1,2,13,18]. This nonplanar steroid
activates both the human and the rodent PXR at super-
physiological concentrations, but has a preferential affinity
Correspondence to J. Uppenberg, Structural Chemistry, Biovitrum,
Lindhagensgatan 133, S-112 76, Stockholm, Sweden.
Fax: + 46 86972320, Tel.: + 46 86973136,
E-mail:
Abbreviations: PCN, pregnenolone-16a-carbonitrile; LBD, ligand
binding domain.
*Note: these authors contributed equally to this work.
Present address: Neuronova AB, Fiskartorpsva
¨
gen 15 A, S-114 33,
Stockholm, Sweden.
àPresent address: KaroBio AB, Novum, S-141 57 Huddinge, Sweden.
(Received 19 June 2002, revised 8 August 2002,
accepted 28 August 2002)
Eur. J. Biochem. 269, 4896–4904 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03207.x
for the rodent receptors [13]. The species-specific induction
pattern of PXR is possibly an adaptive response to the
environment and a need to adjust toxicological responses to
endogenously produced substances. A transgenic mouse
over-expressing the human PXR has been developed with

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

mutagenesis Kit (Clontech). The following primers
were used: 5¢-TCGAGCTGTGTATACTGAGATTCA-3¢
for Q285I, 5¢-TCAATGCTCAGCAGACCCAGCGGC-3¢
for H407Q, 5¢-TCAATGCTCAGGCCACCCAGCG
GC-3¢ for H407A. The selection restriction site mutation
was created by primer 5¢-GTAGCTGACTGGAGCATG
CAT-3¢ mutating a unique XhoIsite.
Plasmid constructs, mouse PXR
The full-length mouse PXR (mPXR-2) expression vector
was generated by RT-PCR using mouse liver polyA
+ RNA (BalB/c, Clontech). After PCR amplification the
fragment was subcloned into the pcDNA3 vector (Invitro-
gen). Oligonucleotides carrying the amino-acid substitutions
corresponding to the hPXR mutations were designed:
5¢-TGAGATGTGCCAGCTGAGGTTCA-3¢ for I282Q
(forward), 5¢-CAACGCCCAGCATACCCAGCAGT-3¢
for Q404H (forward), 5¢-CAACGCCCAGGCAACCCAG
CAGT-3¢ for Q404A (forward), 5¢-TGAACCTCAGCT
GGCACATCTA-3¢ for I282Q (reverse), 5¢-ACTGCTG
GGTATGCTGGGCGT-3¢ for Q404H (reverse), 5¢-ACT
GCTGGGTTGCCTGGGCGT-3¢ for Q404A (reverse).
Mutations were introduced by PCR mutagenesis in a two
step reaction. The pCDNA3 vector primers used were
na614 5¢-CTGCTTACTGGCTTATCGAA-3¢ (forward)
and na1106 5¢-GGGTCAAGGAAGGCACGG-3¢
(reverse). The mutants were subcloned into the pCDNA3
vector (Invitrogen).
General plasmid constructs
The CYP3A4 luciferase reporter plasmid ()10466 to +53)
has been described previously [9]. The pRSV-AF control

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,

Promega). Cells were harvested and the cell lysates were
analyzed for luciferase activity. All experiments were
performed at least three times in duplicates and luciferase
activity was normalized for alkaline phosphatase activity.
For curve fitting and EC50 calculations,
XLFIT
version 2.0.3
was used.
Western blot analysis
C3A cells were seeded into 75 cm
2
flasks at a density of
3.75 · 10
6
cells per flask and incubated at 37 °Covernight.
Co-transfections were performed as described earlier and
the cells were transfected with 15 lg CYP3A4-luciferase
reporter, 0.75 lg RSV-AF plasmid and 0.375 lg plasmid
containing hPXR/mPXR or a mutant variant thereof. After
24 h the cells were washed and scrapeloaded in NaCl/P
i
.
Cell pellets were collected by centrifugation and resus-
pended in Lysis buffer A (10 m
M
Hepes/KOH pH 7.6,
1.5 m
M
MgCl
2

,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

were removed by centrifugation at 140 00 r.p.m. for 10 min
(4 °C) and the supernatants were saved as nuclear fractions.
The protein content of the nuclear and cytoplasmic
fractions were determined by amino-acid analysis. 250 lL
of 6
M
HCl with 0.5% Phenol was added to each of the
samples (5 lL) and the hydrolysis was carried out at a
temperature of 155 °C for 45 min. For the amino-acid
analysis an AminoQuant II/M High Sensitivity Instrument
(Hewlett Packard, Waldbronn, Germany) was used. The
AminoQuant amino-acid analyzer combines the OPA and
FMOC as derivatization reagents for the complete detection
of all residues. BSA was used as a standard protein to
calculate the amount of protein in the samples. Western blot
analysis was performed using 20 lL(hPXR/mPXR,
respectively) of the nuclear and cytoplasmic fractions mixed
with NuPAGE Sample buffer supplemented with reducing
agent. The mixes were heated at 70 °C for 10 min and the
samples were applied on a 10% NuPAGE Bis/Tris Gel
(Invitrogen). The protein was transferred to a Hybond-C
extra membrane (Amersham Life Science) and blocked in
NaCl/P
i
/Tween supplemented with 5% dry milk overnight.
ThemembranewaswashedwithNaCl/P
i
/Tween and
incubated for 1 h at RT with the hPXR/mPXR specific
antibodies PXR (N-16): sc-9690/PXR (R-14): sc-7739

(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

were modeled as the most likely conformer from the O
structural database. In cases where the side chain modeling
gave rise to close contacts, other energetically favorable
conformations were chosen. The VDR crystal structure [19]
lacks a region of 50 amino acids in the omega-loop that were
deleted in the expression construct to obtain suitable protein
for crystallization. It was suggested that this region lacked
stable structure and therefore interfered with crystallization.
We have consequently not modeled this region of PXR. The
two receptor sequences are furthermore most dissimilar in
this part of the structure. In addition there are four deletions
of one or two amino acids in the LBD of the PXR sequence
as compared to VDR. These are found in surface and loop
regions in the structure and were modeled manually in
O
,
followed by geometric regularization using the refine_zone
command. The model was finally subjected to 50 cycles of
conjugate gradient energy minimization with the program
CNS [21]. The minimized structure was examined for large
structural changes and none were observed. The ligand
binding pocket was identified with the program
VOIDOO
,
using a probe diameter of 1.4 A
˚
[22].
RESULTS
Our homology model of human PXR LBD suggested the
presence of an elongated and closed ligand binding pocket

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

VOIDOO
[22] and represents the surface
accessible by the center of a 1.4-A
˚
probe. The side chains of the two
mutated residues, His407 and Gln285, are both adjacent to the ligand
binding pocket. The crystal structure of human PXR has shown that
these residues are also involved in hydrogen bonding interactions with
the synthetic ligand SR12813 [23].
4898 T. O
¨
stberg et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the full length mouse and human PXR variants, in
combination with a reporter vector containing the CYP3A4
promoter ()10466 to +53) fused to a luciferase reporter
gene. Luciferase activity was measured as read-out after
induction with rifampicin, Pregnenolone-16a-carbonitrile
(PCN) and SR12813 (Fig. 2). All compounds are well
characterized ligands for human and mouse PXR, where
rifampicin and SR12813 are potent activators of the human
receptor and PCN primarily activates the mouse receptor
[1–4,13,18].
During this study a crystal structure of PXR was
published by Watkins et al. [23], which led us to compare
our model with the experimental coordinates (PDB entry
1ILH was used). A total of 204 carbon-alpha atoms with an
rms deviation of 1.50 A
˚
were aligned with the lsq_explicit
option in 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

variants are similar in levels to their corresponding wild-type
human and mouse receptor (Fig. 4). The mutant Q285I in
the human receptor is activated by PCN at lower concen-
trations compared to the wild-type human receptor, with
calculated EC50s of 4 l
M
and 14 l
M
, respectively
(Fig. 5d,F). For rifampicin (Fig. 5A) and SR12813 (Fig. 5c)
we found an approximately twofold decrease in fold
induction by Q285I compared to human wild-type receptor.
The corresponding mutant of the mouse receptor, I282Q,
shows a decreased activation by PCN both in terms of EC50
and fold induction (Fig. 5E). As observed with the wild-type
mouse receptor the I282Q mutant was neither activated by
rifampicin nor SR12813 (data not shown).
Human H407Q and mouse Q404H
The basal activity of the human PXR mutant H407Q is
similar to the wild-type receptor (Fig. 4A). The correspond-
ing mutant of mouse Q404H shows a marked decrease in
basal activity as compared to both the wild-type and the
other mutants of the mouse receptor (Fig. 4B). The human
H407Q mutant and the wild-type receptor are activated to a
similar degree by PCN (Fig. 5D). H407Q is still activated by
rifampicin with a slightly lower EC50, but also with a lower
fold induction (Fig. 5a,F). The SR12813 compound simi-
larly activates H407Q, but with a lower fold induction
(Fig. 5C). The mouse mutant Q404H is strongly activated
by PCN and in terms of fold induction surpasses the wild-

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

hPXR-1. In cells transfected with wild-type or mutant
mPXR a band of similar strength (approximately 50 kDa)
was detected (data not shown, see Discussion). Cytosolic
fractions were also analyzed and only very weak bands
could be detected on a Western blot (data not shown).
DISCUSSION
The PXR nuclear receptor has become a new focus of
nuclear receptor research after the discoveries of its central
role in drug metabolism and xenobiotic signaling. In this
study we have used mutated receptors to investigate the role
of specific residues in receptor activation and in particular
address the different activation profiles observed for human
and mouse PXR. For that purpose we have built a
homology model of human PXR in order to identify
residues that were likely to be involved in ligand binding.
Since the initiation of this study the crystal structure of the
human receptor has been published [23]. This has allowed us
to compare our model with the crystal structure and
validate our choice of mutations. To a large extent our
model corresponds to the crystal structure and our choice of
candidate residues for mutation reflects well the questions
we wanted to address. These residues are also located in
regions where our model agrees closely with the crystal
structure. There are other parts of the model that do not
correlate with the crystal structure, in particular the region
Fig. 4. A diagram of basal transcriptional activity in wild-type and
mutated receptors as determined by a luciferase reporter assay. Prior to
measurements 2 lL DMSO was added to each well. (a) The human
PXR constructs showed similar basal levels with exception for H407A,
which was strongly activated without addition of ligand. (b) The

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

the presence of a truncated protein of a molecular mass
similar to endogenous PXR. This is likely due to an
alternative translation initiation site by a non-AUG
codon [33], which is present in PXR [2]. Any substantial
Fig. 5. Activation curves for different ligands and receptor constructs used in the luciferase reporter assay. Human PXR wild-type and mutant
receptors activated by (a) rifampicin (b) rifampicin (mutant H407A) (c) SR12813 and (d) PCN; (e) mouse PXR wild-type and mutant receptors
activated by PCN. (f) Table of EC50 values as calculated by the program Xlfit.
Ó FEBS 2002 Mutagenesis of PXR ligand binding domain (Eur. J. Biochem. 269) 4901
contribution from the presence of endogenous human PXR
in our experiments is expected to result in a strong induction
by rifampicin or SR12813 in cells transfected with the
mouse PXR. This has not been observed (Fig. 3b).
We also made a Western blot analysis of the mouse
receptor constructs. Although many attempts were made
only weak detection of mouse PXR could be performed
with the antibody at our disposal and we have been unable
to obtain a blot clear enough to print. However we
estimated that the levels of expression are roughly equal
for all constructs.
Mutation of human Gln285 and mouse Ile282
The residue Q285/I282 is located in the ligand binding
pocket on helix 5 with the side chain easily accessible for
potential ligands (Fig. 1). The crystal structure of human
PXR in complex with SR12813 shows how this residue is
involved in hydrogen bonding to the ligand in one of the
three modes that this ligand can bind to the receptor [23].
This is consistent with our mutant Q285I, which has a
slightly decreased ability to be activated by SR12813. This
suggests that at least one of the binding modes of SR12813
have been altered. The activation of Q285I by PCN has

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

a key role in receptor activation. The basal activity in
particular seems to be sensitive to the nature of this residue.
This was evident from the human H407A mutant, where the
basal activity increased dramatically, and the mouse Q404H
mutant, where the basal activity decreased by more than
50% (Fig. 4). The basal activity of H407Q and Q404A on
the other hand remained close to wild-type levels. The
structural reasons for the observed changes in basal activity
are not obvious, although one can speculate on rearrange-
ments of the region around helix 12 (Fig. 1), which is known
to be critical for coactivator binding and thereby activation.
Replacing the histidine with an alanine in the human
receptor creates a void, which is surrounded by the
hydrophobic side chains of Phe281, Met323, Leu411,
Phe420 and Phe429. It is possible that the mutation causes
these side chains to reorient themselves to partly fill this
void. Phe429 is of special interest as it belongs to helix 12
and even a small movement or stabilization of this residue
could be of importance for receptor activation. It is
noteworthy that His407 takes on a different conformation
in the ligand bound structure of PXR, with a side chain
movement away from helix 12, as compared to the apo-
structure. It is interesting to note that a similar mutation in
this area, R410A also creates a constitutively active receptor
[23]. This residue lies side by side with His407 one helical
turn away on helix 11. The replacement of these two large
side chains with the beta-carbon of alanine could introduce
more flexibility to helix 11 itself. Although one cannot
predict exactly what effect this has on the structure, the
proximity to helix 12 both sequentially and geometrically

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].

4902 T. O
¨
stberg et al. (Eur. J. Biochem. 269) Ó FEBS 2002
strong effect of PCN on Q404H should be viewed in
perspective of the basal activity. The full activation of the
mutant is similar in level to the wild-type in absolute terms,
but as the basal activity is lower for the mutant, the number
of fold activation is higher. One can see Q404H as a
sensitized receptor, where the negative effect on the basal
activity is countered and neutralized by the ligand. No
improvement is seen in activation of H407Q by PCN over
wild-type, nor Q404H by rifampicin or SR12813.
Although the mutant H407A shows a high basal activity,
it can be further activated by the potent activator rifampicin.
If the mutation triggers specific conformational changes
that facilitate receptor activation, the binding of ligand may
still improve activation by a general stabilization of the
receptor. This phenomenon has earlier been observed in
NMR studies for the PPARc receptor [24]. No clear
increase in activation by PCN or SR12813 was observed.
SR12813 could be expected to lose some affinity for this
mutant as one of the hydrogen bond partners has been
removed. The mouse mutant Q404A was similar to the
wild-type in its ability to be activated by PCN, while being
unresponsive to rifampicin and SR12813.
The corresponding residue to His407 of human PXR is
remarkably conserved across a wide variety of nuclear
receptors, including the PPARs, TRs, VDRs and RORs
[25]. The crystal structures of these receptors show that this
histidine side chain is interacting directly with ligands and/or

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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