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Molecular Endocrinology, doi:10.1210/me.2006-0323
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Molecular Endocrinology 21 (5): 1028-1038
Copyright © 2007 by The Endocrine Society

Crystal Structure of the Pregnane X Receptor-Estradiol Complex Provides Insights into Endobiotic Recognition

Yu Xue, Linda B. Moore, Jillian Orans, Li Peng, Sompop Bencharit, Steven A. Kliewer and Matthew R. Redinbo

Department of Chemistry (Y.X., J.O., S.B., M.R.R.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; Molecular Discovery Research (L.B.M.), GlaxoSmithKline, Research Triangle Park, North Carolina 27709; Department of Molecular Biology (L.P., S.A.K.), The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390; Department of Biochemistry and Biophysics (M.R.R.), and the Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Matthew R. Redinbo, Ph.D., Department of Chemistry, CB 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 pregnane X receptor (PXR) responds to a wide variety of xenobiotic and endobiotic compounds, including pregnanes, progesterones, corticosterones, lithocholic acids, and 17ß-estradiol. In response to these ligands, the receptor controls the expression of genes central to the metabolism and excretion of potentially harmful chemicals from both exogenous and endogenous sources. Although the structural basis of PXR’s interaction with small and large xenobiotics has been examined, the detailed nature of its binding to endobiotics, including steroid-like ligands, remains unclear. We report the crystal structure of the human PXR ligand-binding domain (LBD) in complex with 17ß-estradiol, a representative steroid ligand, at 2.65 Å resolution. Estradiol is found to occupy only one region of PXR’s expansive ligand-binding pocket, leaving a notable 1000 Å3 of space unoccupied, and to bridge between the key polar residues Ser-247 and Arg-410 in the PXR LBD. Positioning the steroid scaffold in this way allows it to make several direct contacts to {alpha}AF of the receptor’s AF-2 region. The PXR-estradiol complex was compared with that of other nuclear receptors, including the estrogen receptor, in complexes with analogous ligands. It was found that PXR’s placement of the steroid is remarkably distinct relative to other members of the nuclear receptor superfamily. Using the PXR-estradiol complex as a guide, the binding of other steroid- and cholesterol-like molecules was then considered. The results provide detailed insights into the manner in which human PXR responds to a wide range of endobiotic compounds.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PREGNANE X receptor [PXR; also known as steroid and xenobiotic receptor (SXR) and pregnane-activated receptor (PAR)] plays a central role in xenobiotic detection and the subsequent regulation of genes involved in drug metabolism and excretion (1, 2, 3, 4). The receptor has also been shown to respond to key endogenous ligands, including 5-ß-pregnane-3,20-dione, progesterones, corticosterones, testosterone, pregnenolones, lithocholic acids, the steroid-like compound dexamethasone, and 17ß-estradiol (4, 5, 6, 7, 8, 9, 10, 11). Indeed, PXR was termed the pregnane X receptor because it is activated by a variety of C21 steroids (4). In response to these and other chemicals, PXR regulates the expression of gene products involved in protective cholesterol and bile acid metabolism and processing. PXR is a member of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors and, like most NRs, contains both DNA-binding domains and ligand-binding domains (LBDs) connected by a presumably flexible hinge region (12). PXR functions as an obligate heterodimer with the retinoid X receptor and binds to various combinations of direct and everted repeat elements in the regulatory regions of target genes (3). Although it does not contain a lengthy activation function 1 (AF-1) region at its N terminus like other NRs, PXR maintains an intact AF-2 within its LBD, which is stabilized by bound ligand and facilitates the recruitment of transcriptional coactivators (13). Unlike many NRs, however, PXR exhibits a consistent basal transcriptional activity in the absence of ligand (4, 14).

Crystal structures of the PXR LBD have been reported in its apo (unliganded) state, as well as with small drug-like and herbal ligands (e.g. SR12813, hyperforin) and the large macrolide ligand rifampicin (13, 14, 15, 16). These structures have revealed that PXR contains an expansive and structurally conformable ligand-binding pocket capable of changing in shape depending on the nature of its bound ligand. PXR also maintains an approximately 60-residue insert between {alpha}1 and {alpha}3 in the well-established LBD fold, which adds a variety of distinct features to PXR including two ß-strands that extend the standard two- to three-stranded LBD ß-sheet to five strands in PXR (17). The terminal ß-strands in these ß-sheets interact in an ideal antiparallel fashion in PXR to form a homodimer unique to PXR that has recently been shown to be critical for receptor function (18). It has also been noted that the PXR LBD deviates significantly in sequence across species relatively to other NRs, and these differences have led to the hypothesis that the PXRs evolved to respond to xenobiotic or endobiotic pressures distinct to each species (4, 19).

Whereas the nature of PXR’s interactions with xenobiotics has been well examined structurally, no corresponding structural data exist to date on the interaction of PXR with an endogenous ligand, including steroid-like compounds, which appear to be of predominant importance. To address this, we determined the crystal structure of the PXR LBD in complex with 17ß-estradiol, a representative steroid, and refined it to 2.65 Å resolution. This structure reveals that PXR positions estradiol in one hemisphere of its ligand-binding pocket, bridging key polar regions of the receptor and directly contacting the AF-2 region. This binding mode is distinct relative to both the interaction of the estrogen receptor (ER) with the same ligand (20) and the manner in which other NRs contact steroid- or cholesterol-like ligands (21, 22, 23, 24, 25, 26). In addition, the data outlined in this paper support the hypothesis that PXR’s evolution has been significantly impacted by the ability to recognize the bile acids unique to different organisms and to play a protective role in eliminating toxic levels of such compounds. In summary, this structure provides the first scaffold by which the binding of endogenous ligands to PXR can be understood and probed at the molecular level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Overall Structure
To unravel the structural basis of the recognition of endogenous steroid-like ligands by the nuclear xenobiotic receptor PXR, we determined the crystal structure of the PXR LBD in complex with 17ß-estradiol. Repeated attempts to obtain a structure of this complex were hampered by the covalent attachment of the reducing agent dithiothreitol (DTT) to Cys-284 within the ligand-binding pocket of PXR, as visualized within several crystal structures (data not shown). Thus, this cysteine side chain was replaced with serine using PCR mutagenesis, which produced a form of the LBD that generated a stable complex with estradiol. Crystals were obtained, and data were collected to 2.65 Å resolution at the Southeast Regional Collaborative Access Team beamline facility at Advanced Photon Source at Argonne National Labs (Argonne, IL). The structure was determined by molecular replacement and refined using torsion angle dynamics to Rcryst and Rfree values of 0.217 and 0.273, respectively (Table 1Go). The PXR LBD conformation is similar to that observed in other PXR-ligand complexes determined to date (13, 14, 15, 16), exhibiting the three {alpha}-helical layers common to NR LBDs but with the unique PXR features of an extended ß-sheet mediating homodimer formation (Fig. 1AGo). The PXR LBD in the estradiol complex shares 0.5 Å root-mean-square deviation in C{alpha} atom positions with the apo (unliganded) PXR structure (14) and is in the active conformation with regard to its AF-2 surface. Only a few residues adjacent to the ligand-binding pocket exhibit small shifts in position between the two structures, including Ser-247 (60° rotation, producing a ~1-Å shift), Cys-284-Ser (60° rotation, ~2 Å-shift), Leu-411 (rotamer change for 2.5-Å shift), Met-243 (rotamer change for 1.5-Å shift), and Arg-410 (1-Å shift). Thus, the binding of estradiol to the PXR LBD does not induce large structural changes relative to the unliganded form of the receptor.


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  		Table 1. Crystallographic Statistics for the PXR-Estradiol Complex

 

Figure 1
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  		Fig. 1. Crystal Structure of the PXR LBD in Complex with 17ß-Estradiol

A, Crystal structure of the LBD of human PXR (PXR LBD, in red, green, and gray) in complex with 17ß-estradiol (cyan). Note the proximity of the estradiol ligand to {alpha}AF in the AF-2 region of PXR. B, Electron density from an FobsPXR-Est – FobsApo, {phi}calc map at 2.8 Å resolution and contoured at 3{sigma} for 17ß-estradiol within the ligand-binding pocket of PXR. C, 17ß-Estradiol forms hydrogen bonds with Ser-247 and Arg-410 (bold labels) in the PXR ligand-binding pocket, as well as van der Waals contacts with several additional residues. The side chain of Arg-410 is also stabilized by a hydrogen bond with Ser-208. Note that the ligand contacts two residues, Met-425 and Phe-429 (labeled in red), located on {alpha}AF of the PXR AF-2 surface. The view in this figure is nearly normal to the plane of the estrogen ring system and rotated roughly 90° about the vertical axis relative to panel A. D, Stereoview of the superposition between the PXR-estradiol complex (protein in cyan, ligand in green) with the PXR-SR12813 complex (13 ) (protein in purple, ligand in yellow), viewed in the same orientation as in panel C. Hydrogen bonds formed between the ligand and protein side chains are shown in cyan and purple for the estradiol and SR12813 complexes, respectively.

 
Estradiol Binding
The use of FobsLigand – FobsApo, {phi}calc maps to identify clearly the positioning of ligands has been effective in past PXR-agonist complex structure determinations (16). Thus, we calculated an FobsPXR-Est – FobsApo, {phi}calc map at 2.8 Å resolution; clear electron density at 3{sigma} indicated the position of 17ß-estradiol bound within the PXR ligand-binding pocket (Fig. 1BGo). It was particularly helpful to observe density for the extracyclic 18-methyl group, which allowed for unambiguous placement of the ligand in a single orientation. Estradiol refined with relatively high thermal displacement parameters (~85 Å2), although such values have been observed with other ligands bound to PXR (e.g. rifampicin) (15). 17ß-Estradiol forms hydrogen bonds with two PXR side chains and additional interactions with eight other residues. The 3-hydroxyl group on the steroid A-ring forms a 2.7-Å hydrogen bond with Ser-247, whereas the 17ß-hydroxyl group on the D-ring forms a 2.9-Å hydrogen bond with Arg-410 (Fig. 1CGo). Arg-410 is stabilized by a 2.9-Å hydrogen bond with Ser-208, which itself is 3.5 Å from the oxygen of the ligand’s 17ß-hydroxyl group. Arg-410 is further stabilized by a 3.0-Å hydrogen bond with the side chain of Glu-321. The side chain of His-407 was placed in two distinct orientations in this structure, and each orientation forms a 3.3-Å van der Waals contact with atoms within the ligand. Thus, polar interactions appear to position the ligand within one region of the ligand-binding cavity, which leaves a significant portion of the available room within the pocket unoccupied (Fig. 1AGo). The small PXR agonist SR12813 forms some overlapping and some distinct polar interactions (13) relative to those formed by estradiol within the PXR ligand-binding pocket (Fig. 1DGo). For example, both ligands form hydrogen bonds with Ser-247; however, whereas estradiol forms polar contacts with Ser-206 and Arg-410, SR12813 makes a hydrogen bond to His-407. Note that the two serines and His-407 adjust in position by up to 2.7 Å (O{gamma}-O{gamma} in Ser-208) to create the necessary hydrogen bonds with each ligand (Fig. 1DGo).

Nonpolar contacts also play a key role in stabilizing 17ß-estradiol within the ligand-binding pocket of PXR. Phe-420 forms a 4.5-Å edge-to-face contact normal to the conjugated A-ring of the steroid, whereas the side chain of Phe-429 forms a second edge-to-face contact more parallel to the A-ring at a distance of 3.5 Å (Fig. 1CGo). The A-ring is further stabilized by 3.4-Å and 3.6-Å van der Waals contacts with Leu-411 and Met-425, respectively, and by a 3.0-Å interaction between the {pi}-orbitals of Phe-251 and the 3-hydroxyl group of the steroid. The B-ring of the steroid also forms a 3.4-Å van der Waals contact with Met-243. Note that Met-425 and Phe-429, which are directly contacted by estradiol, are located on {alpha}AF of the PXR AF-2 surface; thus, these interactions likely help to stabilize the active AF-2 conformation of the receptor. The volume of the human PXR ligand-binding pocket without estradiol present is 1376 Å (3), as calculated by the method of CASTp (27). In the presence of the ligand, however, this volume only decreases by 395 Å (3), leaving 981 Å (3) unoccupied by ligand.

PXR Mutants
To examine the role that individual residues may play in the activation of PXR by steroid-like compounds, variant forms of the full-length receptor were generated that contain either single-site or double-site mutants. The activation of a luciferase reporter gene under the control of the cytochrome P450–3A4 promoter was examined in CV-1 cells upon treatment with increasing concentrations of the established PXR agonist rifampicin (13, 14, 15), or 17ß-estradiol (Fig. 2Go and Table 2Go). For rifampicin, concentrations between 10 nM and 10 µM were examined. For the weaker agonist estradiol, concentrations up to 100 µM were examined for all receptors so that EC50 values could be calculated (Table 2Go). Wild-type PXR exhibited the moderate basal activation levels commonly observed for PXR (14) and EC50 values of 1.2 and 22 µM in response to rifampicin and estradiol, respectively. The binding affinities of rifampicin and 17ß-estradiol to PXR were measured by scintillation proximity assays to be 5.2 and 5 µM, respectively (data not shown). Three single-site mutations, Ser-208-Ala, Cys-284-Ser, and Arg-410-Leu, produced moderate changes in basal activation levels for PXR [effects seen previously for PXR mutations (14, 15, 16)], but little change in EC50 values for the up-regulation of gene expression by the receptor (Table 2Go and Fig. 2Go). For these mutants, EC50 values for PXR activation were 14–15 µM, relative to 22 µM for wild-type (Table 2Go). Note that the Cys-284-Ser mutation employed to prevent the covalent attachment of DTT during crystallization does not impact the function of the receptor. In contrast to these mutations, the replacement of Ser-247, which also forms a hydrogen bond with estradiol, with alanine significantly impacts the ability of the receptor to respond to this representative steroid ligand (Table 2Go and Fig. 2Go). The EC50 value for estradiol increases from 22 µM for wild-type PXR to more than 100 µM for the Ser-247-Ala variant. For rifampicin, which forms a hydrogen bond with Ser-247 but also forms three additional polar interactions (15), the corresponding EC50 value is not significantly altered (1.2 µM for wild-type vs. 0.56 µM for Ser-247-Ala; Table 2Go). This observation supports the conclusion that a polar contact between Ser-247 and steroid-like endobiotics plays an important role in the activation of gene expression by PXR. The reason that Ser-247 is perhaps more critical than other polar residues in the PXR-estradiol complex is explained by the following observations. Ser-247 is located in close proximity to the AF-2 surface of PXR. For example, it is 3.8 Å and 4.8 Å, respectively, from the side chains of Met-425 and Phe-429, which are directly contacted by estradiol. Indeed, we found that the simultaneous replacement of both of these nonpolar residues with alanine produces a form of the receptor that is completely unresponsive to ligands (Table 2Go and Fig. 2Go).


Figure 2
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  		Fig. 2. Transient Transfections in CV-1 Cells of Wild-Type (WT) and Mutant Forms of Full-Length PXR

Responses of each form of the receptor to rifampicin and 17ß-estradiol were measured. Dose responses for estradiol up to 100 µM were measured for all forms of the receptor.

 

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  		Table 2. EC50 Values for Activation by Rifampicin or 17ß-Estradiol

 
Estradiol Binding by PXR vs. ER
We next sought to compare structurally the binding of estradiol by PXR in relation to the interaction of this steroid hormone to its cognate receptor, the ER, another member of the NR superfamily. The crystal structure of PXR in complex with 17ß-estradiol shares 2.6-Å root mean square deviation over C{alpha} positions with the structure of ER{alpha} (20), and the two LBDs share 16% sequence identity. A superposition of the LBDs reveal that, whereas the overall folds of the proteins are similar, the positions of the bound ligands within the pockets are distinct (Fig. 3Go). Estradiol in PXR binds closely adjacent to {alpha}AF and leaves a portion of the large ligand-binding pocket in this receptor unoccupied. In contrast, estradiol is more centrally located in the ER{alpha} pocket, is oriented nearly perpendicular to that observed in the PXR complex, and occupies all but approximately 56 Å3 of the space in the central pocket. ER{alpha} forms three hydrogen bonding and 12 nonpolar contacts with estradiol, whereas PXR forms only two hydrogen bonds and eight van der Waals interactions (Table 3Go). The 3-hydroxyl group on the A-ring of the steroid forms two hydrogen bonds in the ER{alpha} complex, with Glu-353 and Arg-394; in PXR, these residues are replaced with the hydrophobic residues Met-250 and Val-291, respectively (Fig. 3Go). The A-ring is further contacted in ER{alpha} by an edge-to-face interaction with Phe-404, which is replaced by Cys-301 in PXR. Thus, contacts analogous to those observed between ER{alpha} and the A-ring of estradiol are not possible in PXR. ER{alpha} forms a hydrogen bond between His-524 and the 17-hydroxyl group in estradiol; His-524 is replaced by Arg-410 in PXR, which forms a similar interaction in the PXR-estradiol complex. The D-ring is further contacted by Met-421 in ER{alpha}, which is equivalent to Ser-208 in PXR. In summary, only one of three hydrogen bonds formed by ER{alpha} with estradiol is conserved in PXR, and only three of 12 van der Waals contacts are maintained (Table 3Go). Indeed, key residues involved in PXR’s contact with estradiol are replaced by significantly different side chains in ER{alpha} as well. For example, the PXR residues Ser-247, His-407, Phe-420, Phe-429, and Phe-251 are equivalent to alanine, glycine, valine, and two leucine residues, respectively, that do not form analogous contacts in ER{alpha} (Fig. 3Go; Table 3Go). Significantly, however, both complexes with estradiol reveal that direct contacts are formed between the ligand and amino acid side chains on the {alpha}AF helices within the AF-2 region (Met-425 in PXR, Leu-540 in ER{alpha}), providing a molecular explanation for the ability of these agonists to facilitate gene activation by the receptors.


Figure 3
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  		Fig. 3. Stereoview of Ligand-Binding Pockets of PXR and ER{alpha} (red and green, respectively) and the Interactions They Make with 17ß-Estradiol (cyan and yellow, respectively)

The view is the same as that in Fig. 1AGo.

 

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  		Table 3. Comparison of Amino Acids Lining the PXR and ER{alpha} Ligand-Binding Pockets

 
Insights into PXR Activation by Other Endogenous Ligands
PXR is well established as both a xenobiotic and endobiotic sensor. Endogenous ligands known to activate human PXR-mediated gene expression include pregnanes, progesterone, corticosterone, testosterone, lithocholic acids, and the steroid-like xenobiotic dexamethasone (1, 4, 5, 7, 8, 9, 11). We next sought to examine the basis of PXR activation by endogenous compounds by using as a guide the PXR-estradiol structure, the first determined for a steroid scaffold ligand in complex with this receptor. As summarized above, estradiol bridges between two polar regions within the PXR pocket, represented by Ser-247 and Arg-410 (Fig. 4AGo). Arg-410 is also proximal to the surface of the receptor and to a series of other polar side chains that are either adjacent to (e.g. Ser-206 at 3.5 Å) or more distant from estradiol, such as Arg-413, Asp-205, Lys-204, and Asp-230 (Fig. 4AGo). Using binding modes analogous to estradiol as a guide, a series of endogenous compounds that are known PXR activators were modeled into the receptor’s ligand-binding pocket. Significantly, all compounds capable of maintaining contacts to Ser-247 and the Arg-410 regions were established PXR agonists. For example, 5-ß-pregnane-3,20-dione, a ligand for which PXR (the pregnane-X-receptor) is named (4), is capable of forming hydrogen bonds with both Ser-247 and Arg-410. Indeed, modeling this ligand into the PXR pocket using the estradiol structure as a guide reveals that hydrogen bonds to both side chains are likely (Fig. 4BGo). Additional ligands with 3-keto groups, regardless of the conjugated state of their A-rings, also appeared capable of receiving hydrogen bonds from Ser-247 (Fig. 4AGo). In the cases of corticosterone, cortisol, 3-keto-lithocholic acid, and 3-keto-7{alpha},12{alpha}-dihydroxy-5{alpha}-cholanic acid, additional favorable contacts were also likely. For example, the polar side chain of His-407 appeared ideally positioned to interact with hydroxyl or keto oxygens at either the 11 or 12 positions on the steroid scaffolds (see corticosterone, cortisol, and 3-keto-7{alpha},12{alpha}-dihydroxy-5{alpha}-cholanic acid in Fig. 4AGo). Modeling 3-keto-lithocholic acid into the PXR pocket using the estradiol binding mode as a guide reveals expected hydrogen bonds to Ser-247, Arg-410, and Glu-321 (Fig. 4CGo). In addition, extended side chains at the 17 positions of the steroid are also observed to make favorable contacts with polar residues adjacent to Arg-410, such as those shown for dexamethasone-t-butylacetate and 3-keto-7{alpha},12{alpha}-dihydroxy-5{alpha}-cholanic acid. 3ß-Acetate moieties were also found to be capable of forming polar interactions with Ser-247, which explains the activation of PXR by lithocholic acid acetate and lithocholic acid acetate methyl ester (Fig. 4AGo). Similarly, 3ß- and 3{alpha}-hydroxyl groups appeared appropriately positioned to hydrogen bond to Ser-247, helping to explain the agonist character of pregnenolones and dehydroepiandrosterone (Fig. 4AGo). Certain bile acids identified as activators of human PXR were also found to be capable of spanning the distance between Ser-247 and the polar residues adjacent to Arg-410, including glycolithocholic acid and taurolithocholic acid. Taken together, the data presented here provide a structural framework in which endogenous, steroid-like molecules may be understood at the molecular level to be ligands for human PXR.


Figure 4
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  		Fig. 4. PXR Ligand-Binding Pocket with Modeled 3-Keto-Lithocholic Acid and 5-ß-Pregnane-3,20-Dione

A, Analysis of the potential interactions between PXR and other endogenous ligands (as well as dexamethasone) predicted by the PXR-estradiol complex reported here. A schematic view of the proximity of key side chains adjacent to estradiol is shown at top (S247, R410, S208), along with several residues near the 17-OH moiety. Known PXR agonists with 3-keto, 3-ß-acetate, 3-ß-hydroxy, and 3-{alpha}-hydroxy groups are all predicted to form favorable interactions with S247; as shown, further favorable interactions may be formed with additional PXR residues, including H407, R413, D205, and K204. B, The endogenous PXR activator 5-ß-pregnane-3,20-dione (magenta), modeled into the PXR ligand-binding pocket (red) using PXR-estradiol complex as a guide and viewed in the roughly same orientation as Fig. 3Go, may form polar interactions with Ser-247 and Arg-410. C, The endogenous PXR activator 3-keto-lithocholic acid (cyan), modeled into the PXR ligand-binding pocket (red) using PXR-estradiol complex as a guide and viewed in the roughly same orientation as Fig. 3Go, may form polar interactions with Ser-247, Glu-321, and Arg-410.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The human PXR LBD, which has been shown to respond to a variety of both endogenous and xenobiotic compounds, shares structural similarity with the NR superfamily LBDs (28), including that of ER. However, in this report, we show that PXR binds to ER’s endogenous ligand, 17ß-estradiol, in a manner markedly distinct from the estrogen receptor. Although estradiol fills the ligand-binding pocket of ER, it binds adjacent to the {alpha}AF of the AF-2 region in PXR, leaving a significant portion of PXR’s large binding pocket unoccupied. Several structural features within each receptor lead to this difference in binding orientation. The central ß-strand (ß2) in ER’s three-stranded ß-sheet dips deeper into the ligand-binding pocket in this receptor relative to PXR, and positions Phe-404 such that it can form an edge-to-face contact with the bound estradiol molecule (20). ER also contains a longer {alpha}6 and no equivalent to the {alpha}2- and ß1/ ß1'-secondary structural elements of PXR; together, these features allow ER to place a large number of side chains snugly within the ligand-binding pocket to contact estradiol. In addition, although PXR’s pocket is more expansive than that of ER, key estradiol-contacting residues in ER are replaced in PXR. For example, Glu-353 and Arg-394 of ER correspond to Met-250 and Val-291 in PXR, respectively, neither of which is capable of forming central polar contacts to the steroid ligand. Phe-288 and His-407 in PXR, which replace Leu-391 and Gly-521 in ER, respectively, would also clash at approximately 1 Å from estradiol bound as it is observed in ER{alpha}. Taken together, these observations indicate that key structural differences in the LBD folds of ER and PXR allow these two NRs to bind 17ß-estradiol in remarkably distinct manners, revealing the plasticity present in the common NR fold.

Several other NRs bind to steroid-like ligands, including the glucocorticoid receptor (GR), the liver X receptor (LXR), and the constitutive androstane receptor (CAR). We compared the PXR-estradiol complex structure with those of GR-dexamethasone (21), LXR-25-epoxycholesterol (25), and CAR-5ß-pregnane-3,20-dione (26) (Fig. 5Go). Similar to ER-estradiol, the GR and LXR complexes reveal that the steroid ligand is bound nearly perpendicular to that observed in PXR. The contacts leading to these binding modes are similar to that described above for ER{alpha}, including the packing of several side chains into the ligand-binding pockets of GR and LXR to generate receptor-ligand interactions. Recall that GR and LXR are both highly specific for steroid-like ligands, whereas PXR exhibits a much broader ligand-binding profile. CAR, another receptor that responds somewhat promiscuously to ligands, including estradiol (45), positions 5ß-pregnane-3,20-dione in a manner similar to the placement of estradiol in PXR. However, in CAR, 5ß-pregnane-3,20-dione does not directly contact {alpha}AF of the receptor’s AF-2 domain; instead, Asn-165 and Tyr-326, which interact with the ligand, mediate contacts to {alpha}AF (26). In PXR, these residues are replaced by Ser-247 and His-407, respectively, which form both key interactions with estradiol and also facilitate the positioning of the ligand in direct contact with {alpha}AF. The farnesoid X receptor (FXR), which responds to bile acids, was shown previously to orient 6-ethyl-chenodeoxylchlolic acid similar to that observed for dexamethasone-bound GR, with the distinction that the A-ring faces the opposite direction in FXR (22). Again, this position is distinct relative to the observed estradiol docking adjacent to {alpha}AF in the PXR complex presented here; indeed, the position of the loop after {alpha}1 in PXR, which deviates by up to 9 Å relative to the same region in FXR, makes the analogous interaction of bile acids to PXR impossible (data not shown). Taken together, these observations highlight the largely distinct nature of PXR’s interaction with steroid-like ligands relative to other members of the NR superfamily.


Figure 5
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  		Fig. 5. Superpositions of the PXR-Estradiol Structure on that of the GR-Dexamethasone (DEX) Complex (Top), the LXR-25-Epoxycholesterol (eChol) Complex (Middle), and the CAR-5ß-Pregnane-3,20-Dione (Pregnane) Complex (Bottom)

The view is the same as that shown in Fig. 3Go and focusing on the ligand-binding pockets of the receptors.

 
It has been proposed that bile acids played a key role in driving the evolution of the PXRs in a variety of organisms (8). Indeed, PXR has been shown to respond to toxic levels of bile acids and to coordinate the expression of protective gene products capable of metabolizing and excreting bile acids that may accumulate in hepatocytes (5, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38). Schuetz and colleagues (8) recently examined the activation by bile acids of a wide variety of PXRs and PXR-like receptors, including those from human, mouse, rat, dog, cow, pig, rhesus, rabbit, chicken, the zebrafish PXR, and Xenopus benzoate receptor. They concluded that, in terms of responses to endogenous bile acids present in each organism, the receptors could be grouped in the following fashion: human; dog, pig, rhesus (and, likely, cow); mouse and rat; rabbit; chicken X receptor; benzoate X receptor; zebrafish PXR. Using the human PXR-estradiol complex as a guide, we examined the conservation of key residues in the human PXR ligand binding across these orthologous receptors (Table 4Go). We found that as the receptors deviate in their responses to distinct bile acids, the conservation of central residues within the binding pocket clearly diminishes. Thus, our structural data support the conclusion that differences in the nature of endogenous bile acids may have played a significant role in the evolution of the PXRs and PXR-like receptors in a wide variety of organisms.


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  		Table 4. Comparison of Ligand-Binding Pocket Amino Acids among NRs Related to PXR

 
In summary, the data presented here reveal that PXR interacts with the steroid-like molecules by positioning them such that they contact Ser-247, Arg-410, and {alpha}AF of the receptor’s AF-2 surface. As noted in Fig. 2Go, PXR has a relatively robust basal activation level, which is then significantly enhanced in the presence of ligands. The binding of a ligand like estradiol with a moderate EC50 in close proximity to the AF-2 surface likely allows PXR to enhance transcriptional activation without the need for high ligand binding affinity. The placement of steroid-scaffold in PXR is distinct relative to other NR-steroid interactions, including that of GR, LXR, and the CAR receptor, which shares some overlapping biological functions with PXR. This structure provides a framework by which the interactions of other endogenous steroid- and cholesterol-like molecules can be understood, and their potential physiological roles probed both in vitro and in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Protein Expression and Purification
The PXR LBD expression construct was engineered as an N-terminal polyhistidine-tagged fusion protein with residues 130–434 from the human PXR. The fusion insert was subcloned into the pRSETA expression vector (Invitrogen, Carlsbad, CA). Cys-284 within the PXR ligand-binding pocket was mutated to serine, to prevent oxidation with DTT during crystallization, using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The mutant was confirmed by sequence analysis. Residues 623–710 of the human steroid receptor coactivator 1(SRC-1) gene were subcloned into the bacterial vector pACYC184 along with a T7 promoter (39). The PXR LBD/pRSETA and the SRC-1/pACYC184 plasmids were cotransformed into the BL21(DE3) strain of Escherichia coli. Shake flask liquid cultures (10 liters) containing standard Luria-Bertani (LB) broth with 0.1 mg/ml ampicillin and 0.034 mg/ml chloramphenicol were inoculated and grown at 22 C for 20 h. The cells were harvested by centrifugation (20 min, 3500 x g, 4 C), and the cell pellet was stored at –80 C. The cell pellet was resuspended in 100 ml Buffer A (50 mM Tris-Cl, pH 7.8; 250 mM NaCl; 50 mM Imidazole, pH 7.5; 5% glycerol). Cells were sonicated for 3–5 min on ice, and the cell debris was removed by centrifugation (90 min, 20,000 x g, 4 C). The cleared supernatant was loaded on to a 50-ml ProBond nickel-chelating resin (Invitrogen). After washing to baseline with Buffer A, the column was washed with Buffer B containing 75 mM imidazole (pH 7.5) and Buffer C containing 75 mM imidazole (pH 7.5) and 50 mM NaCl. The PXR LBD/SRC-1 complex was eluted from the column using Buffer D with 250 mM imidazole (pH 7.5) and NaCl 50 mM. Column fractions were pooled and subjected to SP cation exchange column (Bio-Rad Laboratories, Inc., Hercules, CA) preequilibrated with buffer containing 20 mM Tris-Cl (pH 7.8), 50 mM NaCl, 5 mM DTT, 2.5 mM EDTA (pH 8.0), 5% glycerol. The column was washed to baseline with the same buffer and fractions containing the PXR/SRC-1 complex were eluted at 400 mM NaCl and pooled and diluted 2-fold with the dilution buffer (20 mM Tris-Cl, pH 7.8; 5 mM DTT; 2.5 mM EDTA, pH 8.0; 5% glycerol). The protein was concentrated using Centri-prep 30K (Millipore Corp., Billerica, MA) units.

Crystallization
The human PXR LBD/SRC-1 complex (hPXR-LBD/SRC-1) was concentrated in the presence of 100-fold molar excesses of 17ß-estradiol to a final concentration of 4 mg/ml. Crystallization was achieved by hanging-drop vapor diffusion against the following conditions at 22 C: 50 mM imidazole at pH 7.1, 10% 2-propanol, (vol/vol).

Data Collection and Structure Determination
The structure of the estradiol-bound form of the LBD of human PXR was determined by molecular replacement using the crystal structure of the apo (unliganded) PXR as a search model (14). Rotation and translation function searches were performed using AMoRe (40); clear solutions for each were obtained in the proper space group, P43212. The structure was refined using the torsion angle protocol in crystallography and NMR system with the maximum likelihood function as a target and included an overall anisotropic B-factor and a bulk solvent correction (41). Of the observed data, 10% were set aside for cross-validation using the free-R statistic before any structural refinement (42). Manual adjustments and rebuilding of the model were performed using O (43) and {sigma}A-weighted electron density maps (44). The structure exhibits good geometry (Table 1Go) with no Ramachandran outliers.

Transient Transfections
Mutant forms of full-length PXR were generated using the QuikChange mutagenesis kit (Stratagene) according to the manufacturer’s instructions. All mutants were confirmed by sequence analysis. Transient transfection and reporter gene assays were performed as described previously (14, 15, 16). 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 secreted placental alkaline phosphatase 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 (14, 15, 16). Transfections were performed with LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) essentially according to the manufacturer’s instructions. Drug dilutions of estradiol (Sigma, St. Louis, MO) 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, St. Louis, MO) 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.


   FOOTNOTES
 
This work was supported by The Robert A. Welch Foundation (to S.A.K.) and the National Institutes of Health Grant DK62229 (to M.R.R).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 27, 2007

Abbreviations: AF-2, Activation function 2; CAR, constitutive androstane receptor; DTT, dithiothreitol; ER, estrogen receptor; FXR, farnesoid X receptor; GR, glucocorticoid receptor; LBD, ligand-binding domain; LXR, liver X receptor; NR, nuclear receptor; PXR, pregnane X receptor; SRC, steroid receptor coactivator.

Received for publication August 8, 2006. Accepted for publication February 19, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. 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[Abstract/Free Full Text]
  2. Blumberg B, Sabbagh Jr W, Juguilon H, Bolado Jr J, van Meter CM, Ong ES, Evans RM 1998 SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev 12:3195–3205[Abstract/Free Full Text]
  3. Goodwin B, Redinbo MR, Kliewer SA 2002 Regulation of cyp3a gene transcription by the pregnane x receptor. Annu Rev Pharmacol Toxicol 42:1–23[CrossRef][Medline]
  4. Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, Perlmann T, Lehmann JM 1998 An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92:73–82[CrossRef][Medline]
  5. Goodwin B, Gauthier KC, Umetani M, Watson MA, Lochansky MI, Collins JL, Leitersdorf E, Mangelsdorf DJ, Kliewer SA, Repa JJ 2003 Identification of bile acid precursors as endogenous ligands for the nuclear xenobiotic pregnane X receptor. Proc Natl Acad Sci USA 100:223–228[Abstract/Free Full Text]
  6. 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[Abstract/Free Full Text]
  7. Jones SA, Moore LB, Shenk JL, Wisely GB, Hamilton GA, McKee DD, Tomkinson NC, LeCluyse EL, Lambert MH, Willson TM, Kliewer SA, Moore JT 2000 The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol Endocrinol 14:27–39[Abstract/Free Full Text]
  8. Krasowski MD, Yasuda K, Hagey LR, Schuetz EG 2005 Evolution of the pregnane X receptor: adaptation to cross-species differences in biliary bile salts. Mol Endocrinol 19:1720–1739[Abstract/Free Full Text]
  9. Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA 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[Medline]
  10. Moore LB, Goodwin B, Jones SA, Wisely GB, Serabjit-Singh CJ, Willson TM, Collins JL, Kliewer SA 2000 St. John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc Natl Acad Sci USA 97:7500–7502[Abstract/Free Full Text]
  11. Moore LB, Parks DJ, Jones SA, Bledsoe RK, Consler TG, Stimmel JB, Goodwin B, Liddle C, Blanchard SG, Willson TM, Collins JL, Kliewer SA 2000 Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J Biol Chem 275:15122–15127[Abstract/Free Full Text]
  12. Carnahan VE, Redinbo MR 2005 Structure and function of the human nuclear xenobiotic receptor PXR. Curr Drug Metab 6:357–367[Medline]
  13. Watkins RE, Davis-Searles PR, Lambert MH, Redinbo MR 2003 Coactivator binding promotes the specific interaction between ligand and the pregnane X receptor. J Mol Biol 331:815–828[CrossRef][Medline]
  14. Watkins RE, Wisely GB, Moore LB, Collins JL, Lambert MH, Williams SP, Willson TM, Kliewer SA, Redinbo MR 2001 The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science 292:2329–2333[Abstract/Free Full Text]
  15. Chrencik JE, Orans J, Moore LB, Xue Y, Peng L, Collins JL, Wisely GB, Lambert MH, Kliewer SA, Redinbo MR 2005 Structural disorder in the complex of human pregnane X receptor and the macrolide antibiotic rifampicin. Mol Endocrinol 19:1125–1134[Abstract/Free Full Text]
  16. Watkins RE, Maglich JM, Moore LB, Wisely GB, Noble SM, Davis-Searles PR, Lambert MH, Kliewer SA, Redinbo MR 2003 2.1 A crystal structure of human PXR in complex with the St. John’s wort compound hyperforin. Biochemistry 42:1430–1438[CrossRef][Medline]
  17. Orans J, Teotico DG, Redinbo MR 2005 The nuclear xenobiotic receptor pregnane X receptor: recent insights and new challenges. Mol Endocrinol 19:2891–2900[Abstract/Free Full Text]
  18. Noble SM, Carnahan VE, Moore LB, Luntz T, Wang H, Ittoop OR, Stimmel JB, Davis-Searles PR, Watkins RE, Wisely GB, Lecluyse E, Tripathy A, McDonnell DP, Redinbo MR 2006 Human PXR forms a tryptophan zipper-mediated homodimer. Biochemistry 45:8579–8589[CrossRef][Medline]
  19. Kliewer SA, Goodwin B, Willson TM 2002 The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr Rev 23:687–702[Abstract/Free Full Text]
  20. Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson J-A, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  21. Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ, McKee DD, Consler TG, Parks DJ, Stewart EL, Willson TM, Lambert MH, Moore JT, Pearce KH, Xu HE 2002 Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 110:93–105[CrossRef][Medline]
  22. Mi LZ, Devarakonda S, Harp JM, Han Q, Pellicciari R, Willson TM, Khorasanizadeh S, Rastinejad F 2003 Structural basis for bile acid binding and activation of the nuclear receptor FXR. Mol Cell 11:1093–1100[CrossRef][Medline]
  23. Shan L, Vincent J, Brunzelle JS, Dussault I, Lin M, Ianculescu I, Sherman MA, Forman BM, Fernandez EJ 2004 Structure of the murine constitutive androstane receptor complexed to androstenol: a molecular basis for inverse agonism. Mol Cell 16:907–917[Medline]
  24. Suino K, Peng L, Reynolds R, Li Y, Cha JY, Repa JJ, Kliewer SA, Xu HE 2004 The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization. Mol Cell 16:893–905[Medline]
  25. Williams S, Bledsoe RK, Collins JL, Boggs S, Lambert MH, Miller AB, Moore J, McKee DD, Moore L, Nichols J, Parks D, Watson M, Wisely B, Willson TM 2003 X-ray crystal structure of the liver X receptor ß ligand binding domain: regulation by a histidine-tryptophan switch. J Biol Chem 278:27138–27143[Abstract/Free Full Text]
  26. Xu RX, Lambert MH, Wisely BB, Warren EN, Weinert EE, Waitt GM, Williams JD, Collins JL, Moore LB, Willson TM, Moore JT 2004 A structural basis for constitutive activity in the human CAR/RXR{alpha} heterodimer. Mol Cell 16:919–928[CrossRef][Medline]
  27. Binkowski TA, Naghibzadeh S, Liang J 2003 CASTp: computed atlas of surface topography of proteins. Nucleic Acids Res 31:3352–3355[Abstract/Free Full Text]
  28. Ingraham HA, Redinbo MR 2005 Orphan nuclear receptors adopted by crystallography. Curr Opin Struct Biol 15:708–715[CrossRef][Medline]
  29. Frank C, Makkonen H, Dunlop TW, Matilainen M, Vaisanen S, Carlberg C 2005 Identification of pregnane X receptor binding sites in the regulatory regions of genes involved in bile acid homeostasis. J Mol Biol 346:505–519[CrossRef][Medline]
  30. Guo GL, Lambert G, Negishi M, Ward JM, Brewer Jr HB, Kliewer SA, Gonzalez FJ, Sinal CJ 2003 Complementary roles of farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor in protection against bile acid toxicity. J Biol Chem 278:45062–45071[Abstract/Free Full Text]
  31. Kliewer SA, Willson TM 2002 Regulation of xenobiotic and bile acid metabolism by the nuclear pregnane X receptor. J Lipid Res 43:359–364[Abstract/Free Full Text]
  32. Sonoda J, Chong LW, Downes M, Barish GD, Coulter S, Liddle C, Lee CH, Evans RM 2005 Pregnane X receptor prevents hepatorenal toxicity from cholesterol metabolites. Proc Natl Acad Sci USA 102:2198–2203[Abstract/Free Full Text]
  33. Staudinger J, Liu Y, Madan A, Habeebu S, Klaassen CD 2001 Coordinate regulation of xenobiotic and bile acid homeostasis by pregnane X receptor. Drug Metab Dispos 29:1467–1472[Abstract/Free Full Text]
  34. Staudinger JL, Goodwin B, Jones SA, Hawkins-Brown D, MacKenzie KI, Latour A, Liu Y, Klaassen CD, Brown KK, Reinhard J, Willson TM, Koller BH, Kliewer SA 2001 The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity. Proc Natl Acad Sci USA 98:3369–3374[Abstract/Free Full Text]
  35. Stedman CA, Liddle C, Coulter SA, Sonoda J, Alvarez JG, Moore DD, Evans RM, Downes M 2005 Nuclear receptors constitutive androstane receptor and pregnane X receptor ameliorate cholestatic liver injury. Proc Natl Acad Sci USA 102:2063–2068[Abstract/Free Full Text]
  36. Uppal H, Toma D, Saini SP, Ren S, Jones TJ, Xie W 2005 Combined loss of orphan receptors PXR and CAR heightens sensitivity to toxic bile acids in mice. Hepatology 41:168–176[CrossRef][Medline]
  37. Xie W, Radominska-Pandya A, Shi Y, Simon CM, Nelson MC, Ong ES, Waxman DJ, Evans RM 2001 An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids. Proc Natl Acad Sci USA 98:3375–3380[Abstract/Free Full Text]
  38. Zhang J, Huang W, Qatanani M, Evans RM, Moore DD 2004 The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid-induced hepatotoxicity. J Biol Chem 279:49517–49522[Abstract/Free Full Text]
  39. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-{gamma}. Nature 395:137–143[CrossRef][Medline]
  40. Navaza J, Saludjian P 1997 AMoRe: an automated molecular replacement package. Methods Enzymol 276A:581–594
  41. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL 1998 Crystallography, NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921[CrossRef][Medline]
  42. Brünger AT 1993 Assessment of phase accuracy by cross validation: the free R value. Methods and applications. Acta Crystallogr D Biol Crystallogr 49:24–36[CrossRef][Medline]
  43. Jones TA, Zou JY, Cowan SW, Kjeldgaard M 1991 Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47:110–119[CrossRef]
  44. Read RJ 1986 Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr A 42:140–149[CrossRef]
  45. Kawamoto T, Kakizaki S, Yoshinari K, Negishi M 2000 Estrogen activation of nuclear orphan receptor CAR (constitutive active receptor) in induction of the mouse CYP2b10 gene. Mol Endocrinol 14:1897–1905[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   LXRβ  |  LXRα  |  PXR  |  CAR  |  GR
Coregulators:   SRC-1
Ligands:   Dexamethasone  |  Hydrocortisone  |  17β-Estradiol  |  Progesterone



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