title: Construction and characterization of a fully active PXR/SRC-1 tethered protein with increased stability.
abstract: The nuclear xenobiotic receptor PXR is a ligand-inducible transcription factor regulating drug-metabolizing enzymes and transporters and a master switch mediating potentially adverse drug-drug interactions. In addition to binding a coactivator protein such as SRC-1, the C-terminal ligand-binding domain (LBD) is solely responsible for ligand recognition and thus the ligand-dependent downstream effects. In an effort to facilitate structural studies of PXR to understand and abolish the interactions between PXR and its ligands, several recombinant PXR/SRC-1 constructs were designed and evaluated for expression, stability and activity. Expression strategies employing either dual expression or translationally coupled bicistronic expression were found to be unsuitable for producing stable PXR in a stochiometric complex with a peptide derived from SRC-1 (SRC-1p). A single polypeptide chain encompassing PXR and SRC-1p tethered with a peptidyl linker was designed to promote intramolecular complex formation. This tethered protein was overexpressed as a soluble protein and required no additional SRC-1p for further stabilization. X-ray crystal structures in the presence and absence of the known PXR agonist SR-12813 were determined to high resolution. In addition, a circular dichroism-based binding assay was developed to allow rapid evaluation of PXR ligand affinity, making this tethered protein a convenient and effective reagent for the rational attenuation of drug-induced PXR-mediated metabolism.
authors.affiliation: Structural Chemistry Department, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA. wenyan.wang@spcorp.com
authors.forename: Wenyan
authors.initials: W
authors.lastname: Wang
authors.forename: Winifred W
authors.initials: WW
authors.lastname: Prosise
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: S Shane
authors.initials: SS
authors.lastname: Taremi
authors.forename: Hung V
authors.initials: HV
authors.lastname: Le
authors.forename: Vincent
authors.initials: V
authors.lastname: Madison
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Cui
authors.forename: Ann
authors.initials: A
authors.lastname: Thomas
authors.forename: K-C
authors.initials: KC
authors.lastname: Cheng
authors.forename: Charles A
authors.initials: CA
authors.lastname: Lesburg
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D022781:Hepatocytes / Q000378:metabolism; D051548:Histone Acetyltransferases / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D056920:Nuclear Receptor Coactivator 1; D000077297:Pregnane X Receptor; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods; D011987:Receptors, Steroid / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1