Coactivator Binding Promotes the Specific Interaction Between Ligand and the Pregnane X Receptor

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Abstract

The pregnane X receptor (PXR) detects the presence of a wide variety of endogenous and xenobiotic compounds, and is a master regulator of the expression of genes central to drug metabolism and excretion. We present the 2.0 Å crystal structure of the human PXR ligand-binding domain (LBD) in complex with the cholesterol-lowering compound SR12813 and a 25 amino acid residue fragment of the human steroid receptor coactivator-1 (SRC-1) containing one LXXLL motif. PXR crystallizes as a homodimer in the asymmetric unit in this structure and possesses a novel α2 helix adjacent to its ligand-binding cavity. The SRC-1 peptide forms two distinct helices and binds adjacent to the ligand-dependent transactivation AF-2 helix on the surface of PXR. In contrast with previous PXR structures, in which SR12813 bound in multiple orientations, the small SR12813 agonist in this structure binds in a single, unique orientation within the receptor's ligand-binding pocket and contacts the AF-2 helix. Thermal denaturation studies reveal that the SR12813 ligand and SRC-1 coactivator peptide each stabilize the LBD of PXR, and that together they exert an additive effect on the stability of the receptor. These results indicate that the binding of coactivator to the surface of PXR limits the ability of this promiscuous receptor to “breathe” and helps to trap a single, active conformation of SR12813. They further reveal that specificity is required for PXR activation.

Introduction

The human pregnane X receptor (PXR) is a nuclear xenobiotic receptor that detects potentially toxic chemicals and regulates the expression of genes central to their breakdown and removal. The large and growing array of genes regulated by PXR correspond to all phases of drug metabolism, including those encoding cytochrome P450 (CYP) alleles 3A, 2C, and 1A, glutathione-S-transferase enzymes, and the drug efflux pumps MDR1 (multi-drug resistance gene-1; P-glycoprotein) and MDR2.1., 2., 3., 4., 5., 6., 7. PXR acts promiscuously and detects a wide variety of structurally distinct xenobiotics (foreign chemicals) and endobiotics (endogenous chemicals).1., 5., 6., 8., 9., 10., 11., 12., 13., 14., 15., 16., 17., 18., 19. Drugs known to activate PXR range from the small barbituate phenobarbital (232 Da) to the much larger antibiotic rifampicin (823 Da) and the anticancer drug taxol (854 Da).5., 17. PXR appears to work in concert with the less promiscuous constitutive androstane receptor (CAR) to regulate the expression of an overlapping set of genes involved in xenobiotic metabolism and excretion.20., 21., 22., 23.

The activation of PXR can lead to a dangerous class of drug–drug interactions. Patients taking the unregulated herbal antidepressant St. John's wort along with oral contraceptives, cancer chemotherapeutics, the immunosuppressant cyclosporin, or the anti-HIV drug indinavir have exhibited significant reductions in their serum levels of these critical compounds.24., 25., 26., 27., 28., 29. Hyperforin, the active agent of St. John's wort, was shown to be a high-affinity ligand for human PXR (Kd 27 nM).30., 31. Hyperforin binds to PXR and induces the expression of a wide variety of drug metabolism and efflux genes in primary human hepatocytes, including CYP3A4,1., 6., 32., 33., 34., 35. responsible for breaking down greater than 50% of known drugs,36 and the efflux pump MDR-1.2., 5., 35. Thus, the activation of PXR by compounds like hyperforin can lead to the unwanted metabolism and excretion of other important clinical drugs.

The structure of the ligand-binding domain (LBD) of human PXR has been examined crystallographically both in its apo form (without ligand), in complex with the cholesterol-lowering compound SR12813 (Kd 41 nM),37 and in complex with St. John's wort compound hyperforin.35 The PXR LBD has an unusually large and structurally conformable ligand-binding cavity.35., 37., 38. While the structure of the PXR-hyperforin complex revealed this high-affinity ligand bound to PXR in a single orientation,35 the small SR12813 ligand was present in three distinct orientations in the PXR–SR12813 complex. Thus, the flexible pocket of PXR may allow the binding of structurally diverse ligands in one or more orientations.

Nuclear receptors regulate gene expression by binding to target DNA sequences in the regulatory regions of genes, and by recruiting transcriptional coactivators and corepressors in a ligand-dependent manner. Most nuclear receptors bind to DNA as dimers. The class I steroid hormone receptors, such as the estrogen (ER) and androgen (AR) receptors, function as homodimers, while class II nuclear receptors, like PXR, act as a heterodimers with RXRα.6 PXR binds to the p160/SRC (steroid receptor coactivator) family of coactivators including SRC-1,6 which interacts with histone acetyltransferase (HAT) complexes, CBP (CREB-binding protein)/p300,39., 40. and contains a weak HAT activity of its own at its carboxyl terminus.41 SRC-1 recruits the arginine methyltransferase CARM-1,42 which methlyates both histone H343 and CBP/p300, and helps to shift the pool of nuclear CBP/p300 proteins toward exclusive interactions with nuclear receptors.44

The p160/SRC family of coactivators are characterized by three LXXLL (where L is Leu and X is any amino acid) motifs that bind to the active conformation of the AF-2 (ligand-dependent activating function) helix on nuclear receptor LBDs.45., 46., 47., 48., 49. The highly mobile AF-2 helices are shifted in position, depending on whether ligands, transcriptional coactivators or corepressors are bound.45., 46., 47., 50., 51., 52., 53., 54., 55., 56., 57. Crystal structures of nuclear receptors bound to fragments of the transcriptional coactivator SRC-145., 46., 47., 52. have revealed that the LXXLL motif also adopts an α-helical conformation and binds to the surface of nuclear receptors at a cleft formed, in part, by the AF-2 helix. Main-chain atoms of the LXXLL helix interact with two conserved charged residues on the surface of the nuclear receptor, forming a “charge clamp” that is observed in many coactivator–nuclear receptor interactions.45., 46., 47., 51., 52., 53., 54., 55., 56.

We present the crystal structure of the PXR LBD bound to both the SR12813 agonist and a 25 amino acid residue fragment of human SRC-1. The SRC-1 peptide is stabilized on the surface of PXR by a charge clamp, and forms a second short helix perpendicular to the LXXLL helix. The binding of SRC-1 has marked effects on the manner in which the SR12813 agonist interacts with PXR. SR12813 is bound to the PXR–coactivator complex in a single orientation that is distinct from the three orientations observed previously37 and contacts the AF-2 helix directly. CD-thermal denaturation studies of the PXR LBD reveal that both SR12813 and the SRC-1 peptide stabilize PXR and that the effects of this stabilization are additive. Thus, the binding of a coactivator to PXR appears to limit the ability of the receptor to “breathe”, helping to trap an active orientation of SR12813. Specific interactions between ligands and PXR also appear to be required for PXR activation.

Section snippets

Overall structure of the PXR/SR12813/SRC-1 complex

We determined the crystal structure of the human PXR LBD bound to a peptide of human SRC-1 (residues 676–700) and the cholesterol-lowering compound SR12813 at 2.0 Å resolution. This complex crystallized in space group P212121 with a homodimer in the asymmetric unit (Figure 1; Table 1). All PXR LBD crystals obtained previously were of space group P43212 and contained one molecule in the asymmetric unit;35., 37. however, the PXR LBD molecules formed a homodimer related by crystallographic symmetry

Discussion

We present and examine the structure of the LBD of the human nuclear xenobiotic receptor PXR in a ternary complex with an LXXLL-containing peptide of the transcriptional coactivator SRC-1 and the cholesterol-lowering compound SR12813. This is the first structure of PXR in complex with a region of a transcriptional coregulatory protein. The PXR LBD forms a novel homodimer in the asymmetric unit of this structure (Figure 1). We are currently examining the possible functional role of this

Protein purification and crystallization

The human PXR LBD (residues 130–434) was coexpressed in Escherichia coli BL21(DE3) cells with an 88 amino acid fragment of SRC-1 (residues 623–710), and purified by Ni-affinity chromatography as described.37 The SRC-1 fragment contains two LXXLL motifs, is required for the stable expression of PXR, and remains bound to PXR throughout purification. The SRC-1 fragment is lost, however, during crystallization in this and previous PXR structures, as confirmed by SDS-PAGE analysis of carefully

Acknowledgements

The authors thank Steve Kliewer, Tim Willson, and Bruce Wisely, as well as members of the Redinbo Laboratory for thoughtful discussions and experimental assistance. This work was funded by the NIH (R01-DK62229).

References (84)

  • S.C. Piscitelli et al.

    Indinavir concentrations and St John's wort

    Lancet

    (2000)
  • F. Ruschitzka et al.

    Acute heart transplant rejection due to Saint John's wort

    Lancet

    (2000)
  • G. Bertilsson et al.

    Functionally conserved xenobiotic responsive enhancer in cytochrome P450 3A7

    Biochem. Biophys. Res. Commun.

    (2001)
  • Y. Kamei et al.

    A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors

    Cell

    (1996)
  • R.T. Gampe et al.

    Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors

    Mol. Cell

    (2000)
  • H. Greschik et al.

    Structural and functional evidence for ligand-independent transcriptional activation by the estrogen-related receptor 3

    Mol. Cell

    (2002)
  • A.K. Shiau et al.

    The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen

    Cell

    (1998)
  • P. Cronet et al.

    Structure of the PPARalpha and -gamma ligand binding domain in complex with AZ 242; ligand selectivity and agonist activation in the PPAR family

    Structure (Camb)

    (2001)
  • P.M. Matias et al.

    Structural evidence for ligand specificity in the binding domain of the human androgen receptor. Implications for pathogenic gene mutations

    J. Biol. Chem.

    (2000)
  • N. Rochel et al.

    The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand

    Mol. Cell

    (2000)
  • H.E. Xu et al.

    Molecular recognition of fatty acids by peroxisome proliferator-activated receptors

    Mol. Cell

    (1999)
  • W. Bourguet et al.

    Crystal structure of a heterodimeric complex of RAR and RXR ligand-binding domains

    Mol. Cell

    (2000)
  • J.D. Love et al.

    The structural basis for the specificity of retinoid-X receptor-selective agonists: new insights into the role of helix H12

    J. Biol. Chem.

    (2002)
  • D.M. Heery et al.

    Core LXXLL motif sequences in CREB-binding protein, SRC1, and RIP140 define affinity and selectivity for steroid and retinoid receptors

    J. Biol. Chem.

    (2001)
  • R.K. Bledsoe et al.

    Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition

    Cell

    (2002)
  • V.H. Coulthard et al.

    An extended LXXLL motif sequence determines the nuclear receptor binding specificity of TRAP220

    J. Biol. Chem.

    (2003)
  • P. Pissios et al.

    Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding

    Mol. Cell

    (2000)
  • B.A. Johnson et al.

    Ligand-induced stabilization of PPARgamma monitored by NMR spectroscopy: implications for nuclear receptor activation

    J. Mol. Biol.

    (2000)
  • J. Navaza et al.

    AMoRe: an automated molecular replacement package

    Methods Enzymol.

    (1997)
  • Z. Otwinowski et al.

    Processing of X-ray diffraction data collected in oscillation mode

    Methods Enzymol.

    (1997)
  • J.M. Lehmann et al.

    The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions

    J. Clin. Invest.

    (1998)
  • S. Gerbal-Chaloin et al.

    Induction of CYP2C genes in human hepatocytes in primary culture

    Drug. Metab. Dispos.

    (2001)
  • T.W. Synold et al.

    The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux

    Nature Med.

    (2001)
  • J.M. Rosenfeld et al.

    Genetic profiling defines the xenobiotic gene network controlled by the nuclear receptor PXR

    Mol. Endocrinol.

    (2003)
  • A. Takeshita et al.

    Bisphenol-A, an environmental estrogen, activates the human orphan nuclear receptor, steroid and xenobiotic receptor-mediated transcription

    Eur. J. Endocrinol.

    (2001)
  • H. Masuyama et al.

    Endocrine disrupting chemicals, phthalic acid and nonylphenol, activate pregnane X receptor-mediated transcription

    Mol. Endocrinol.

    (2000)
  • C. Handschin et al.

    Conservation of signaling pathways of xenobiotic-sensing orphan nuclear receptors, chicken xenobiotic receptor, constitutive androstane receptor, and pregnane X receptor, from birds to humans

    Mol. Endocrinol.

    (2001)
  • P.B. Desai et al.

    Induction of cytochrome P450 3A4 in primary human hepatocytes and activation of the human pregnane X receptor by tamoxifen and 4-hydroxytamoxifen

    Drug. Metab. Dispos.

    (2002)
  • L. Drocourt et al.

    Calcium channel modulators of the dihydropyridine family are human pregnane X receptor activators and inducers of cyp3a, cyp2b, and cyp2c in human hepatocytes

    Drug. Metab. Dispos.

    (2001)
  • M.C. Wright

    The cytochrome P450 3A4 inducer metyrapone is an activator of the human pregnane X receptor

    Biochem. Soc. Trans.

    (1999)
  • E.G. Schuetz et al.

    Environmental xenobiotics and the antihormones cyproterone acetate and spironolactone use the nuclear hormone pregnenolone X receptor to activate the CYP3A23 hormone response element

    Mol. Pharmacol.

    (1998)
  • B. Goodwin et al.

    The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module

    Mol. Pharmacol.

    (1999)
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    Present address: P. R. Davis-Searles, RTI International, 3040 Cornwallis Road, P.O. Box 12194, Research Triangle Park, NC 27709, USA.

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