Structural Disorder in the Complex of Human Pregnane X Receptor and the Macrolide Antibiotic Rifampicin

Jill E. Chrencik, Jillian Orans, Linda B. Moore, Yu Xue, Li Peng, Jon L. Collins, G. Bruce Wisely, Millard H. Lambert, Steven A. Kliewer and Matthew R. Redinbo

Department of Chemistry (J.E.C., J.O., Y.X., M.R.R.) and Department of Biochemistry & Biophysics, Program in Molecular Biology and Biotechnology, and the Lineberger Comprehensive Cancer Center (M.R.R.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; Nuclear Receptor Discovery Research (L.B.M., J.L.C., G.B.W., M.H.L.), GlaxoSmithKline, Research Triangle Park, North Carolina 27709; and Department of Molecular Biology (L.P., S.A.K.), The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390

Address all correspondence and requests for reprints to: Matthew R. Redinbo, Ph.D, Department of Chemistry, Campus Box 3290, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290. E-mail: redinbo{at}unc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human nuclear xenobiotic receptor, pregnane X receptor (PXR), detects a variety of structurally distinct endogenous and xenobiotic compounds and controls expression of genes central to drug and cholesterol metabolism. The macrolide antibiotic rifampicin, a front-line treatment for tuberculosis, is an established PXR agonist and, at 823 Da, is one of the largest known ligands for the receptor. We present the 2.8 Å crystal structure of the ligand-binding domain of human PXR in complex with rifampicin. We also use structural and mutagenesis data to examine the origins of the directed promiscuity exhibited by the PXRs across species. Three structurally flexible loops adjacent to the ligand-binding pocket of PXR are disordered in this crystal structure, including the 200–210 region that is part of a sequence insert novel to the promiscuous PXRs relative to other members of the nuclear receptor superfamily. The 4-methyl-1-piperazinyl ring of rifampicin, which would lie adjacent to the disordered protein regions, is also disordered and not observed in the structure. Taken together, our results indicate that one wall of the PXR ligand-binding cavity can remain flexible even when the receptor is in complex with an activating ligand. These observations highlight the key role that structural flexibility plays in PXR’s promiscuous response to xenobiotics.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HUMAN NUCLEAR xenobiotic receptor, pregnane X receptor (PXR), controls the expression of genes involved in xenobiotic metabolism and cholesterol homeostasis (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). PXR up-regulates the expression of cytochrome P450 3A4 (CYP3A4), which metabolizes more than 50% of human drugs, in response to xenobiotics, but down-regulates CYP7A1 expression in response to toxic bile acids (6, 8, 12). PXR is activated by an array of structurally distinct endogenous and xenobiotic compounds that vary in size from small compounds such as phenobarbital (232 Da) to large drugs like the macrocyclic antibiotic rifampicin (823 Da; Fig. 1Go) (5, 6, 13, 14). The increase in expression of drug metabolism genes mediated by PXR can cause dangerous drug-drug interactions by enhancing the metabolism of other drugs such as oral contraceptives, antivirals, or immunosuppressants (11, 15, 16).



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Fig. 1. Electron Density for Rifampicin

A, Chemical structure of rifampicin with the 4-methyl-1-piperazinyl ring for which there is no clear electron density indicated in gray. Positions of specific atoms identified in panels B and C are labeled. B, Stereoview of |FRifampicinobs| – |FApoobs| electron density at 2.8 Å resolution and contoured at 1{sigma} of the ligand-binding pocket of PXR. Rifampicin was placed into this density at an initial round of structure refinement. C, Stereoview of 2|Fobs| – |Fcalc| electron density at 2.8 Å resolution and contoured at 1{sigma} for rifampicin in the ligand-binding pocket after structural refinement.

 
Crystal structures of the human PXR ligand-binding domain (LBD) have revealed the three-layered {alpha}-helical sandwich typical for nuclear receptors (17, 18), but with features that appear key to the ligand binding promiscuity of the receptor (11, 16, 19). PXR contains an insert of approximately 60 amino acids not found in the primary sequence of other nuclear receptors. This insert creates a novel, extended five-stranded antiparallel ß-sheet in PXR and a 13- to 20-amino acid stretch of disordered residues adjacent to the ligand-binding pocket. Typical nuclear receptor LBDs contain a two- to three-stranded antiparallel ß-sheet (17). These features in PXR combine to generate a flexible and conformable ligand-binding pocket that adjusts its shape to accommodate ligands of distinct size and structure. Twenty-eight amino acid side chains line PXR’s pocket, of which eight are polar and capable of forming hydrogen bonds with ligands (16). All ligands examined to date form a combination of hydrophobic and polar interactions with PXR ligand-binding pocket residues.

PXRs from different species exhibit directed promiscuity and respond to partially overlapping sets of ligands from distinct regions of chemical space (16). For example, the human and rabbit PXRs are strongly activated by rifampicin whereas the mouse and rat PXRs are not; the mouse and rat PXRs, in contrast, are activated by PCN (pregnenolone 16{alpha}-carbonitrile) whereas human PXR is not (13, 20, 21, 22). It has been shown that a small number of amino acid changes within the ligand-binding pocket can alter the ability of particular ligands to activate specific mammalian PXRs (22). These observations have led to the hypothesis that the directed promiscuity exhibited by PXRs across species arose due to distinct xenobiotic and endobiotic pressures exerted on species during evolution.

We present the 2.8 Å resolution crystal structure of the human PXR LBD in complex with the macrolide antibiotic rifampicin, which is used to treat tuberculosis (23, 24, 25). Rifampicin is an established PXR agonist and potent activator of CYP3A4 expression in humans (6) and is one of the largest PXR ligands identified to date. The PXR-rifampicin structure reveals that the macrocyclic ring of rifampicin fits within the PXR ligand-binding pocket, whereas the drug’s 4-methyl-1-piperazinyl ring and several protein loops are disordered. These observations suggest that PXR responds to large ligands by using structural flexibility to enhance the effective size of its ligand-binding pocket, and that certain regions of the pocket may not require structure for transcription to be activated by the receptor. The structure also provides insights into the selective response to rifampicin exhibited by PXRs from different species.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rifampicin Binding Induces Structural Disorder in PXR
The 2.8 Å resolution crystal structure of the human PXR LBD in complex with rifampicin reveals an LBD-like fold for PXR, consisting of a three-layered {alpha}-helical sandwich, but with the five-stranded antiparallel ß-sheet unique to this receptor (16) (Fig. 2Go). The LBD in this complex is similar to the apo-, SR12813-, and hyperforin-bound PXR structures determined previously, with root-mean-square deviations of 0.56, 0.80, and 0.86 Å, respectively, over 300 equivalent C{alpha} positions (11, 16). Three regions located adjacent to the ligand-binding pocket at the bottom of the LBD, however, were disordered and exhibited no clear electron density: residues 178–209, 229–235, and 310–317. Amino acids 178 to 191–197 have been disordered in all PXR LBD structures examined to date (11, 16, 19). The remaining regions disordered in the PXR-rifampicin structure were observed in previous structures, although they exhibited relatively high thermal displacement parameters (crystallographic B factors) that indicate structural mobility.



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Fig. 2. Crystal Structure of the LBD of Human PXR ({alpha}-Helices in Cyan, ß-Strands in Yellow) in Complex with Rifampicin (Red) at 2.8 Å Resolution

The PXR-LBD consists of a three-layered {alpha}-helical sandwich with a five-stranded antiparallel ß-sheet. The following loop regions of PXR are disordered in this complex: residues 178–209, 229–235, and 310–317.

 
Rifampicin was placed in a single orientation within the ligand-binding pocket using 2.8 Å resolution |FobsRif| – |FobsApo|, {Phi}calc and |Fobs| – | Fcalc|, {Phi}calc electron density maps (Fig. 1Go). The position of the conjugated napthalene ring with associated furane was evident in these initial difference maps, as was placement of more than half the atoms in the macrolide ring of the drug. Ordered electron density for two regions of the compound, however, were not present in these initial maps: the 4-methyl-1-piperazinyl ring and an adjacent 4-carbon aliphatic portion of the macrolide ring (Fig. 1Go). Convincing electron density for these regions of the drug did not appear during structural refinement; thus, they are not present in the final model of the complex. Liquid chromatography/mass spectrometry was performed on PXR-rifampicin complex crystals dissolved after extensive washing, and this analysis confirmed the presence of intact rifampicin, not a breakdown product, within the crystals (data not shown). The 4-methyl-1-piperazinyl ring and adjacent 4-carbon aliphatic region of rifampicin’s macrolide ring are in proximity of disordered regions of PXR in this structure, which helps to explain their mobility and lack of clear electron density.

Rifampicin contacts 18 amino acid side chains in the PXR ligand-binding pocket, including four hydrogen bonding contacts (Fig. 3Go). SR12813 and hyperforin, in contrast, contacted 11 and 13 residues, respectively, in PXR complexes with these compounds reported previously. Rifampicin forms hydrogen bonds with Ser-247 and His-407 and two hydrogen bonds with Gln-285; these residues have been observed to make polar contacts with SR12813 and with hyperforin as well (11, 16, 19). Rifampicin interacts with several residues, including Val-211, Leu-239, Leu-308, and Arg-410, not contacted directly in previous PXR complexes (Fig. 3Go). Interactions with these residues are generated by the size of the rifampicin ligand, which is 40% larger than hyperforin and SR12813 and occupies regions of the binding pocket not filled by these smaller ligands.



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Fig. 3. Stereoview of the Contacts Made by Rifampicin (Yellow) with Amino Acid Side Chains (Green) that Line the PXR Ligand Binding Cavity, Viewed in the Same Orientation as in Fig. 2Go.

 
The regions of PXR that are disordered in this structure cluster together at the bottom of the LBD (Fig. 4Go). In Fig. 4Go, these loops have been modeled as they appear in the structure of the unliganded PXR LBD structure reported previously (16). Two of these regions (178–209, 229–235) are on an approximately 60-amino acid stretch (~175–235) that is unique to the PXRs of known sequence and have not been found in other members of the nuclear receptor superfamily. Although amino acids 178 to 191–197 have been disordered in all PXR LBD structures examined to date, the pseudohelical region that starts between residues 192 and 198 and ends at residue 209 has been observed in previous structures. This pseudohelical region has been found to change its position to accommodate the binding of different ligands (11, 16, 19). The 310–317 loop forms a novel {alpha}-helix 6 that leads into {alpha}-helix 7 when this region is ordered. It appears that these three regions (200–209, 229–235, and 310–317), which cluster together at the bottom of the PXR ligand-binding cavity, work together to create a flexible floor to the receptor’s ligand-binding cavity. Such a mobile floor is structurally unique to the PXRs and appears critical to the promiscuous ligand-binding character of this xenobiotic sensor.



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Fig. 4. Crystallographic Thermal Displacement Parameters (B Factors) for Ordered Regions within the PXR-Rifampicin Complex Are Indicated via Color Ramping from Blue (Relatively Low; 20 Å2) to Red (Relatively High; ≤90 Å2)

Disordered regions of the structure (see Fig. 2Go), with thermal displacement parameters more than 90 Å2, are indicated in gray as they appear in the structure of unliganded PXR (16 ). Amino acids 178 to approximately 200 have been disordered in all PXR LBD structures examined to date. The view in this figure is rotated approximately 180° about the vertical axis relative to Fig. 2Go.

 
Rifampicin Binding Examined by Mutagenesis
Targeted mutations of amino acid side chains that line the pocket of human PXR were introduced to examine the importance of individual contacts between rifampicin and PXR in transient transfection assays. The small and well-studied PXR agonist SR12813 was used as a control. Wild-type human PXR exhibited EC50 values of 700 (±130) nM and 140 (±38) nM for rifampicin and SR12813, respectively (Fig. 6AGo). Mutation of either Gln-285 to isoleucine or His-407 to glutamine did not impact by more than 3-fold the EC50 value of rifampicin for PXR (Fig. 5Go and Fig. 6Go, B and C). This was particularly surprising for Gln-285, which forms two hydrogen bonds with rifampicin; mutation to isoleucine would eliminate such polar interactions. Thus, hydrogen bonding at this position is apparently not required for receptor activation by rifampicin. In support of this conclusion, rabbit PXR, which is more potently activated by rifampicin than human PXR, contains a leucine at the equivalent position. The alteration of His-407 to glutamine moderately increases the basal (ligand-independent) activation level of this mutant form of the receptor relative to wild type.



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Fig. 6. Dose-Response Curves from Transient Transfection Experiments for Wild-Type and Mutant Forms of PXR in Response to Rifampicin (Black) and SR12813 (Blue), a Control PXR Agonist

Mutants presented in these panels correspond to those shown in Fig. 5Go. EC50 values accompany curves for which reliable values could be obtained.

 


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Fig. 5. Mutations and Amino Acid Changes around the PXR Ligand-Binding Pocket in the PXR-Rifampicin Complex (Green) Considered in this Paper

Human PXR residues are in magenta and site-directed mutants examined are in cyan. At amino acid position 308, the valine side chain found in rabbit PXR is in light blue, and the phenylalanine side chain found in mouse and rat PXR is in blue.

 
Mutation of Arg-410, which forms a van der Waals contact with rifampicin, to asparagine reduces by approximately 3-fold the activation of the receptor by this macrolide (Figs. 5Go and 6DGo). This mutation also lowers the EC50 of SR12813 by about 2-fold. The contact between Arg-410 and rifampicin is apparently important for the activation of human PXR. It may also be that the salt bridge formed between Arg-410 and Glu-321 is critical for the receptor’s response to ligands. The mutation of Asp-205, which forms a salt bridge with Arg-413 in structures determined previously, to alanine improves by approximately 3-fold the activation of PXR by rifampicin, but worsens the EC50 value of SR12813 by about 3-fold and eliminates the basal activity of PXR (Figs. 5Go and 6EGo). It is likely that for small ligands such as SR12813, the ability of Asp-205 to form an ionic interaction with Arg-413 helps to close the receptor around the ligand. In the case of rifampicin, however, the elimination of this salt bridge may provide additional room or flexibility in the binding pocket to allow this large ligand to bind.

Alterations of Ser-247 to tryptophan or of Trp-299 to alanine largely eliminate PXR’s response to rifampicin or SR12813 (Fig. 5Go and Fig. 6Go, F and G). Ser-247 and Trp-299 form hydrogen bonds or hydrophobic contacts, respectively, with all ligands examined to date (11, 16, 19). Receptors containing these single-site mutations exhibited no basal repression of transcriptional activation and instead appear to be constitutively active. The replacement of the smaller serine side chain with tryptophan at position 247 likely sterically blocks ligand binding and partially fills the binding pocket, possibly explaining the ligand-independent activation of this mutant receptor. The fact that the elimination of the large tryptophan side chain at position 299 has the same effect on receptor activity is more difficult to understand. Two possible explanations are that this mutant form of the receptor is compromised in its ability to bind corepressor or is enhanced in its ability to bind coactivator. Similar effects on basal repression have been seen with mutations at other positions in PXR (16).

Species-Specific Activation of PXR by Rifampicin
PXRs from different species exhibit directed promiscuity. For example, rifampicin activates rabbit PXR more effectively than human PXR, but is a poor agonist for both rat and mouse PXR. Changing the amino acid at position 308 in PXR was reported to impart sensitivity to rifampicin using scanning mutagenesis in a chimeric rat/human receptor (22). Leu-308, which is a phenylalanine in rat and mouse PXR, forms a 3.4 Å nonpolar contact with rifampicin in the structure presented here. Our crystal structure suggested that replacement of this leucine with phenylalanine would result in a steric clash with both the ligand and the adjacent indole ring of Trp-299 (Figs. 3Go and 5Go). To test this hypothesis, we generated a Phe-305-Val mutant form of mouse PXR (305 is the 308 equivalent in the mouse receptor) and examined the action of this full-length variant receptor in transient transfection assays. Valine was chosen because it is the side chain observed in this position in rabbit PXR, the receptor isoform for which rifampicin is the most effective ligand. As shown in Fig. 7Go, wild-type human PXR responds well to rifampicin (but not to the control mouse agonist 5-pregnen-3ß-ol-20-one-16{alpha}-carbonitrile, PCN), whereas wild-type mouse PXR does not respond well to rifampicin. A Phe-305-Val form of mouse PXR does gain a weakly (~3-fold) improved response to rifampicin, whereas its response to PCN is reduced by nearly 100-fold. These results indicate that the small side chain in the position equivalent to 308 in human PXR is required to accommodate rifampicin in the receptor’s ligand-binding pocket, as expected from our crystal structure. In addition, these data suggest that the larger phenylalanine side chain at position 305 in mouse PXR is involved in the productive binding to PCN.



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Fig. 7. Dose-Response Curves for Wild-Type (WT) Forms of Human PXR (hPXR) and Mouse PXR (mPXR), and Mutant Forms of mPXR, at Differing Concentrations of Either Rifampicin or the Control Mouse Ligand PCN

Mutant forms of mPXR are indicated using the mouse numbering scheme. EC50 values accompany curves for which reliable values could be obtained.

 
Ser-208, which is located on the disordered 200–210 loop in the structure presented here, is one of eight polar residues that line the human PXR ligand-binding cavity. Human and rabbit PXR contain a serine and threonine, respectively, at this position, whereas rat and mouse PXR contain a much more rigid proline. The proline found in this position in rat and mouse PXR may hinder such flexibility, making the productive binding of rifampicin more difficult. To test this hypothesis, a Pro-205-Ser mutant form of mouse PXR was created and examined in transient transfection assays (205 is the 208 equivalent in mouse PXR). As shown in Fig. 7Go, the Pro-205-Ser mutation did not impart sensitivity to rifampicin on this variant receptor, although this mutation did eliminate the basal transcriptional activity of the receptor. These results indicate that replacing a proline residue at this position with an amino acid that is able to sample more backbone conformational space is not sufficient to impart sensitivity to the large macrolide rifampicin.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rifampicin is widely used to treat tuberculosis and is also employed as a chemoprophylaxis agent for patients exposed to meningococcal disease and Haemophilus influenzae meningitis (23, 24, 25, 27, 28). The macrolide antibiotic is an established human PXR agonist that leads to the potent activation of CYP3A4 expression in primary human hepatocytes (6). The 2.8 Å crystal structure of the human PXR LBD in complex with rifampicin reveals that the macrocyclic ring of the drug fits well within PXR’s ligand-binding pocket. Loops adjacent to the ligand-binding cavity of the receptor, however, and proximal regions of the drug were disordered in this structure (Figs. 1–4GoGoGoGo). Regions that exhibited disorder were novel either in sequence (e.g. amino acids 178–209 and 229–235) or structure (e.g. amino acids 310–317, {alpha}6) to the PXRs. The mobility of these regions, which cluster together at the bottom of the LBD, has been noted in other PXR-ligand complex structures. It appears that PXR has evolved a distinctive and flexible floor to its ligand-binding cavity that allows the receptor to bind to structurally and chemically distinct ligands.

The targeted mutation of amino acids in the ligand-binding pocket of PXR is known to impact the response of the receptor to ligands. We present evidence that some amino acid changes improve the efficacy of rifampicin (Asp-205-Ala), some changes worsen rifampicin efficacy (Arg-410-Asn), and some changes have no effect (Gln-285-Ile) (Figs. 5Go and 6Go). We also present evidence that some amino acid changes impact the basal transcriptional activity of PXR in the absence of ligands (e.g. Asp-205-Ala and Arg-410-Asn exhibited diminished or eliminated basal activity), and in some cases convert the receptor to a ligand-independent transcriptional activator that is constitutively active (Ser-247-Trp, Trp-299-Ala). Similar effects have been seen in previous studies in which mutations were introduced into the PXR ligand-binding pocket (16). The loss of basal transcriptional activity could be caused by changes in the ability of the altered PXRs to interact with transcriptional coregulators. Enhanced corepressor binding or compromised coactivator binding would both lead to decreased basal transcriptional activity. Such effects could be caused by a general destabilization of the PXR structure, or could be specific to particular mutations. The loss of basal repression of transcriptional activity could also be caused by similar factors; for example, mutations may destroy the binding of corepressors or enhance the binding of coactivators. The impact of single mutations on the recruitment of coregulators is an area of future study for PXR and related receptors.

In spite of the ligand binding promiscuity exhibited by PXRs from different species, it is clear that each PXR is also specific to some distinct regions of chemical space. This directed promiscuity may have arisen from evolutionary pressure placed on each species by the potentially harmful xenobiotics and endobiotics they encounter. Rifampicin, for example, is an agonist for human PXR but not for the mouse isoform of the receptor. We found that the replacement of Phe-305 in mouse PXR with valine imparts some sensitivity to rifampicin (Fig. 7Go), an observation that mirrors that presented by Tirona et al. (22). We further found, however, that a Pro-205-Ser mutation in mouse PXR generated a variant form of the rodent receptor that failed to respond to rifampicin (Fig. 7Go). Rifampicin is a more potent agonist for rabbit PXR than it is for human PXR. Subtle chemical changes within the ligand-binding pocket, e.g. replacing Leu-308 in human PXR with the smaller valine in rabbit, appear to play important roles in the directed promiscuity of the PXRs (22). Thus, despite mounting structural data, predicting the responses of particular PXRs to specific ligands remains difficult.

The directed promiscuity exhibited by the PXRs supports the hypothesis that key endogenous or exogenous PXR ligands influenced the evolution of this receptor. For example, the rabbit diet may contain compounds similar to rifampicin in shape or chemical nature, but the human diet may contain less of these compounds and, by extension, the diets of mice and rats contain little of these compounds. Such key evolutionary compounds, however, remain to be confirmed for a PXR. A recent report indicating that human PXR is activated by carotinoids, which are present in micromolar concentrations in human serum, is suggestive in this regard (29).

Indeed, it is possible that such evolutionary ligands are not critical to the directed promiscuity exhibited by the PXRs. An alternative hypothesis is that amino acid changes in the ligand-binding pocket of PXRs from different species arose from random, rather than directed, changes. The following observation can be interpreted to support this conclusion. The sequence identities for 25 amino acids that line the human PXR ligand-binding pocket were calculated relative to the PXRs from six other mammals: rhesus monkey, pig, dog, rabbit, mouse, and rat. The average sequence identity for those ligand-binding, cavity-lining residues was 71%. Sequence identities were also calculated for 25 residues from the surface of PXR (excluding residues involved in known protein-protein interactions) and 25 residues from the hydrophobic core of the PXR LBD fold. The average sequence identities for the surface and core regions of PXR were 75% and 89%, respectively. Thus, residues in the pocket of PXR share a similar average sequence identity across mammalian species to residues on the surface of the receptor. Surface residues are known to change more readily than those in key structural or functional residues, such as in the hydrophobic core of the protein that is required to maintain proper tertiary structure.

These observations could indicate that changes in the ligand-binding pocket residues of PXR across species are random, rather than directed by evolutionary pressure. In this view, the result of evolutionary pressure on the PXRs may simply have been to produce a large and flexible ligand-binding pocket. It is equally possible, however, to interpret these sequence identity observations to indicate that residues in the ligand-binding pocket of the PXRs experienced more evolutionary pressure to change than is typical for amino acids even on the surface of a protein. Such a conclusion supports the directed promiscuity hypothesis for PXR, in which chemical pressure exerted by xenobiotic and endobiotic compounds drove the evolution of a receptor tuned for the ability to detect potentially harmful compounds in each species. In either case, PXR utilizes a large and flexible ligand-binding pocket to detect the presence of a variety of endogenous and xenobiotic compounds, including the macrolide antibiotic rifampicin.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Crystallization and Structure Determination
The PXR LBD (residues 140–434) was coexpressed with a fragment of the steroid receptor coactivator-1 (residues 623–710) in Escherichia coli BL21(DE3) cells and purified using nickel-affinity chromatography as described previously (16). Purified PXR was concentrated to 3 mg/ml in 20 mM Tris-Cl, 250 mM NaCl, 2.5 mM EDTA, 5% glycerol, and a 1000-fold molar excess of rifampicin (Sigma Chemical Co., St. Louis, MO) using an YM-10 spin concentrator (Amicon, Inc., Beverly, MA). The PXR-rifampicin complex was crystallized by hanging-drop vapor diffusion at 25 C against a crystallant of 50 mM imidazole, pH 7.2, and 9% isopropanol, and cryoprotected as described elsewhere (16). Diffraction data were collected at 100 K at the Advanced Photon Source, beamline 22-ID (SER-CAT), and were processed and reduced using HKL2000 (30). The structure was determined by molecular replacement with AMoRE (31) using the structure of apo-PXR [Protein database identification no: 1ILG (16)] as a search model. The structure was refined in Crystallography and NMR System (CNS) using torsion-angle dynamics and the maximum likelihood function target, and manual model adjustments were performed using O (32). Electron density for bound rifampicin was clear in initial |FobsRif| – |FobsApo|, {Phi}calc and |Fobs| – |Fcalc|, {Phi}calc maps at 2.8 Å resolution (Fig. 1Go). However, regions of the structure exhibited poor electron density and thermal displacement parameters (crystallographic B factors) considerably higher than the rest of the structure. Thus, we chose to eliminate from the refinement regions of the protein and ligand that exhibited B factors of more than 90 Å2. The regions that are not present in the refinement are three loops of the protein (residues 178–209, 229–235, and 310–317), and the 4-methyl-1-piperazinyl ring of the ligand. An overall anisotropic B factor and bulk solvent correction were included at the completion of refinement. Final structures exhibit good geometry with no Ramachandran outliers (Table 1Go). Figures were created using Molscript (33), Raster3D (34), Bobscript (35), Dino (www.dino3d.org), and Povray (www.povray.org). The coordinates of the PXR-rifampicin complex, as well as the structure factor data, have been submitted to the Protein Databank at the Research Collaboratory for Structural Bioinformatics (RCSB) and assigned accession code 1SKX.


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Table 1. Crystallographic Analysis of the PXR-Rifampicin Complex

 
Transient Transfection Experiments
Mutations in full-length PXR were generated with the QuikChange Site-Directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Transient transfection and reporter gene assays were performed as described previously (11). CV-1 cells were plated in 96-well plates in phenol red-free DMEM containing high glucose and supplemented with 10% charcoal/dextran-treated fetal bovine serum (HyClone Laboratories, Inc., Logan, UT). Transfection mixes contained 5 ng of receptor expression vector, 20 ng of reporter plasmid, 12 ng of ß-actin secretory placental alkaline phosphatase (SPAP) as internal control, and 43 ng of carrier plasmid. Plasmids for wild-type and mutant forms of human PXR and for the XREM-CYP3A4-LUC reporter, containing the enhancer and promoter of the CYP3A4 gene driving luciferase expression, were as previously described (6, 36). Transfections were performed with LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) essentially according to the manufacturer’s instructions. Drug dilutions of rifampicin (Sigma) and SR12813 (synthesized in house) were prepared in phenol red-free DMEM/F-12 medium with 15 mM HEPES supplemented with 10% charcoal-stripped, delipidated calf serum (Sigma), which had previously been heat inactivated at 62 C for 35 min. Serial drug dilutions were performed in triplicate to generate 11-point concentration response curves. Cells were incubated for 24 h in the presence of drugs, after which the medium was sampled and assayed for alkaline phosphatase activity. Luciferase reporter activity was measured using the LucLite assay system (Packard Instrument Co., Meriden, CT) and normalized to alkaline phosphatase activity. EC50 values were determined by standard methods.


    ACKNOWLEDGMENTS
 
The authors thank Chris Flemming for figure assistance.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK62229 (to M.R.R).

First Published Online February 10, 2005

Abbreviations: CYP3A4, Cytochrome P450 3A4; LBD, Ligand-binding domain; PCN, pregnenolone 16{alpha}-carbonitrile; PXR, pregnane X receptor.

Received for publication September 3, 2004. Accepted for publication January 14, 2005.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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