Journal of Molecular Biology
Coactivator Binding Promotes the Specific Interaction Between Ligand and the Pregnane X Receptor
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).
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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.