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 compare
Trang 1Identification of residues in the PXR ligand binding domain critical for species specific and constitutive activation
Tove O¨stberg1,*, Go¨ran Bertilsson1,*,†, Lena Jendeberg2, Anders Berkenstam2,‡and Jonas Uppenberg3 1
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden;
2
Departments of Biology, and3Structural 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 of PXR
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: jonas.uppenberg@biovitrum.com
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)
Trang 2for 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
M A T E R I A L S A N D M E T H O D S
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¢
GC-3¢ for H407A The selection restriction site mutation
was created by primer 5¢-GTAGCTGACTGGAGCATG
CAT-3¢ mutating a unique XhoI site
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
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)
(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, loti1414101) in 6-well plates C3A cells were seeded at a concentration of 5· 105cells in each well and incubated for 24 h at 37C in 2 mL growth 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 lg RSV-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,XLFITversion 2.0.3 was used
Western blot analysis C3A cells were seeded into 75 cm2 flasks at a density of 3.75· 106cells per flask and incubated at 37C overnight 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/Pi Cell pellets were collected by centrifugation and resus-pended in Lysis buffer A (10 mM Hepes/KOH pH 7.6, 1.5 mM MgCl2, 10 mMKCl, 0.5 mMdithiothreitol, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibi-tors) Nuclear pellets were collected by centrifugation at
4000 r.p.m for 10 min (4C) The supernatants were cleared by centrifugation at 140 00 r.p.m for 10 min (4C) and saved as cytoplasmic fractions The nuclear pellets were resuspended in Lysis buffer B (20 mMHepes/ KOH pH 7.6, 1.5 mMMgCl, 420 mMNaCl, 1 mMEDTA,
Trang 31 mM EGTA, 20% glycerol and protease inhibitors) and
gently mixed for 20 min at 4C The insoluble fractions
were removed by centrifugation at 140 00 r.p.m for 10 min
(4C) 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 6M HCl with 0.5% Phenol was added to each of the
samples (5 lL) and the hydrolysis was carried out at a
temperature of 155C 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 70C 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/Pi/Tween supplemented with 5% dry milk overnight
The membrane was washed with NaCl/Pi/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/Pi/Tween supplemented
with 5% dry milk The membrane was washed with NaCl/
Pi/Tween and subsequently incubated for 45 min with
Peroxidase-conjugated rabbit anti-(goat IgG) Ig (DAKO),
diluted 1/2000 in NaCl/Pi/Tween supplemented with 5%
dry milk A final wash was made with NaCl/Pi/Tween All
NaCl/Pi/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
programO[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 inO,
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]
R E S U L T S
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 · 5 A˚ 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].
Trang 4the 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 programO[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.
Trang 5variants 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 lM and 14 lM, 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 and cells transfected with empty vector, 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)
D I S C U S S I O N
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.
Trang 6neighboring 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.
Trang 7contribution 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.
A weak band detected in the control experiment (lane 1) could be
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].
Trang 8strong 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
A C K N O W L E D G E M E N T S
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.
R E F E R E N C E S
1 Kliewer, S.A., Moore, J.T., Wade, L., Staudinger, J.L., Watson,
M.A., Jones, S.A., McKee, D.D., Oliver, B.B., Willson, T.M.,
Zetterstrom, R.H., Perlmann, T & Lehmann, J.M (1998) An
orphan nuclear receptor activated by pregnanes defines a novel
steroid signaling pathway Cell 92, 73–82.
2 Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg,
L., Sydow-Backman, M., Ohlsson, R., Postlind, H., Blomquist, P.
& Berkenstam, A (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction Proc Natl Acad Sci USA 95, 12208–12213.
3 Blumberg, B., Sabbagh, W Jr, Juguilon, H., Bolado, J Jr, van Meter, C.M., Ong, E.S & Evans, R.M (1998) SXR, a novel steroid and xenobiotic-sensing nuclear receptor Genes Dev 12, 3195–3205.
4 Lehmann, J.M., McKee, D.D., Watson, M.A., Willson, T.M., Moore, J.T & Kliewer, S.A (1998) The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions J Clin Invest 102, 1016–1023.
5 de Wildt, S.N., Kearns, G.L., Leeder, J.S & van den Anker, J.N (1999) Cytochrome P450, 3A: ontogeny and drug disposition Clin Pharmacokinet 37, 485–505.
6 Xie, W., Barwick, J.L., Downes, M., Blumberg, B., Simon, C.M., Nelson, M.C., Neuschwander-Tetri, B.A., Brunt, E.M., Guzelian, P.S & Evans, R.M (2000) Humanized xenobiotic response in mice expressing nuclear receptor SXR Nature 406, 435–439.
7 Goodwin, B., Hodgson, E & Liddle, C (1999) The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module Mol Pharmacol 56, 1329–1339.
8 Pascussi, J.M., Jounaidi, Y., Drocourt, L., Domergue, J., Bala-baud, C., Maurel, P & Vilarem, M.J (1999) Evidence for the presence of a functional pregnane X receptor response element in the CYP3A7 promoter gene Biochem Biophys Res Comm 260, 377–381.
9 Bertilsson, G., Berkenstam, A & Blomquist, P (2001) Function-ally conserved xenobiotic responsive enhancer in cytochrome P450 3A7 Biochem Biophys Res Commun 280, 139–144.
10 Honkakoski, P., Zelko, I., Sueyoshi, T & Negishi, M (1998) The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene Mol Cell Biol 18, 5652–5658.
11 Sueyoshi, T., Kawamoto, T., Zelko, I., Honkakoski, P & Negishi,
M (1999) The repressed nuclear receptor CAR responds to phe-nobarbital in activating the human CYP2B6 gene J Biol Chem.
274, 6043–6046.
12 Wei, P., Zhang, J., EganHafley, M., Liang, S & Moore, D.D (2000) The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism Nature 407, 920–923.
13 Moore, L.B., Parks, D.J., Jones, S.A., Bledsoe, R.K., Consler, T.G., Stimmel, J.B., Goodwin, B., Liddle, C., Blanchard, S.G., Willson, T.M., Collins, J.L & Kliewer, S.A (2000) Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands J Biol Chem 275, 15122–15127.
14 Xie, W., Barwick, J.L., Simon, C.M., Pierce, A.M., Safe, S., Blumberg, B., Guzelian, P.S & M.E.R (2000) Reciprocal acti-vation of xenobiotic response genes by nuclear receptors SXR/ PXR and CAR Genes Dev 14, 3014–3023.
15 Goodwin, B., Moore, L.B., Stoltz, C.M., McKee, D.D & Kliewer, S.A (2001) Regulation of the human CYP2B6 gene by the nuclear pregnane X receptor Mol Pharmacol 60, 427–431.
16 Staudinger, J.L., Goodwin, B., Jones, S.A., Hawkins-Brown, D., MacKenzie, K.I., Latour, A., Liu, Y.P., Klaassen, C.D., Brown, K.K., Reinhard, J., Willson, T.N., Koller, B.H & Kliewer, S.A (2001) The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity Proc Natl Acad Sci USA 98, 3369– 3374.
17 Xie, W., Radominska-Pandya, A., Shi, Y.H., Simon, C.M., Nelson, M.C., Ong, E.S., Waxman, D.J & Evans, R.M (2001) An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids Proc Natl Acad Sci USA 98, 3375–3380.
18 Jones, S.A., Moore, L.B., Shenk, J.L., Wisely, G.B., Hamilton, G.A., McKee, D.D., Tomkinson, N.C., LeCluyse, E.L., Lambert,
Trang 9M.H., Willson, T.M., Kliewer, S.A & Moore, J.T (2000) The
pregnane X receptor: a promiscuous xenobiotic receptor that has
diverged during evolution Mol Endocrinol 14, 27–39.
19 Rochel, N., Wurtz, J.M., Mitschler, A., Klaholz, B & Moras, D.
(2000) The crystal structure of the nuclear receptor for vitamin D
bound to its natural ligand Mol Cell 5, 173–179.
20 Jones, T.A., Zou, J.Y., Cowan, S.W & Kjeldgaard, M (1991)
Improved methods for building protein models in electron density
maps and the location of errors in these models Acta Crystallogr.
A47, 110–119.
21 Bru¨nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros,
P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M.,
Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T & Warren,
G.L (1998) Crystallography and NMR system: a new software
suite for macromolecular structure determination Acta
Crystal-logr D54, 905–921.
22 Kleywegt, G.J & Jones, T.A (1994) Detection, delineation,
measurement and display of cavities in macromolecular structures.
Acta Crystallogr D50, 178–185.
23 Watkins, R.E., Wisely, G.B., Moore, L.B., Collins, J.L., Lambert,
M.H., Williams, S.P., Willson, T.M., Kliewer, S.A & Redinbo,
M.R (2001) The human nuclear xenobiotic receptor PXR:
Structural determinants of directed promiscuity Science 292,
2329–2333.
24 Johnson, B.A., Wilson, E.M., Li, Y., Moller, D.E., Smith, R.G &
Zhou, G (2000) Ligand-induced stabilization of PPARgamma
monitored by NMR spectroscopy: implications for nuclear
receptor activation J Mol Biol 298, 187–194.
25 Wurtz, J.M., Bourguet, W., Renaud, J.P., Vivat, V., Chambon, P.,
Moras, D & Gronemeyer, H (1996) A canonical structure for the
ligand-binding domain of nuclear receptors Nat Struct Biol 3,
87–94.
26 Nolte, R.T., Wisely, G.B., Westin, S., Cobb, J.E., Lambert, M.H., Kurokawa, R., Rosenfeld, M.G., Willson, T.M., Glass, C.K & Milburn, M.V (1998) Ligand binding and co-activator assembly
of the peroxisome proliferator-activated receptor-gamma Nature
395, 137–143.
27 Uppenberg, J., Svensson, C., Jaki, M., Bertilsson, G., Jendeberg,
L & Berkenstam, A (1998) Crystal structure of the ligand binding domain of the human nuclear receptor PPARgamma J Biol Chem 273, 31108–31112.
28 Wagner, R.L., Apriletti, J.W., McGrath, M.E., West, B.L., Baxter, J.D & Fletterick, R.J (1995) A structural role for hormone in the thyroid hormone receptor Nature 378, 690–697.
29 Stehlin, C., Wurtz, J.-M., Steinmetz, A., Greiner, E., Schu¨le, R., Moras, D & Renaud, J.-P (2001) X-ray structure of the orphan nuclear receptor RORb ligand-binding domain in the active con-formation EMBO J 20, 5822–5831.
30 Vivat, V., Zechel, C., Wurtz, J.M., Bourguet, W., Kagechika, H., Umemiya, H., Shudo, K., Moras, D., Gronemeyer, H & Cham-bon, P (1997) A mutation mimicking ligand-induced conforma-tional change yields a constitutive RXR that senses allosteric effects in heterodimers EMBO J 16, 5697–5709.
31 Weis, K.E., Ekena, K., Thomas, J.A., Lazennec, G & Katze-nellenbogen, B.S (1996) Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein Mol Endocrinol 10, 1388–1398.
32 White, R., Sjoberg, M., Kalkhoven, E & Parker, M.G (1997) Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine EMBO J 16, 1427–1435.
33 Kozak, M (1997) Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6 EMBO J 16, 2482–2492.