, External calibration was done in the positive ion mode over the mass range m/z 500-5,000 using the multiply charged ions produced by 0.5 mM horse heart myoglobin solution diluted in water/acetonitrile 50/50 mixture acidified with 0.5% (v/v) formic acid. Purified PXR(130-434)-SRC1 was buffer exchanged against 50 mM ammonium acetate (NH4Ac), pH 8.0 using NAP5 desalting columns (illustra NAP-5 Columns, GE Healthcare Life Sciences), Mass spectrometry experiments were carried out on an electrospray time-of-flight mass spectrometer (LCT, Waters) equipped with an automated chip-based nanoESI device (Triversa Nanomate

, To read a LanthaScreen TR-FRET assay, the fluorimeter (PHERAstar FS; BMG LABTECH) is configured to excite the terbium donor around 340 nm, and to separately read the terbium emission peak that is centred at B490 nm, and the fluorescein emission that is centred at B520 nm. Results are expressed as the signal from the fluorescein emission divided by the terbium signal to provide a TR-FRET emission ratio, Lanthascreen TR-FRET PXR competitive binding assay. GST-hPXR-LBD (10 nM) was incubated with different concentrations

, Protein concentration was determined spectrophotometrically (e 280 nm ¼ 26,210 l mol À 1 cm À 1 ). Duplicate experiments were performed on Microcal ITC200 (Malvern) operating at 25°C. Titrations were carried out in Tris-HCl 20 mM, SRC-1 was dialysed overnight against Tris-HCl 20 mM, pH 8.5, NaCl 200 mM

, Compound solutions were set to 300 mM when tested individually (Fig. 5b,c), 50 mM each when used simultaneously (Fig. 5f) and 50 mM (EE2, Fig. 5d) or 200 mM (TNC, Fig. 5e) when tested after pre-incubation of PXR with 50 mM TNC or EE2, respectively. Heat exchanges were monitored throughout titrations consisting of 19 injections (one time 0.5 ml followed by 18 times 2 ml) of compound solutions into the cell containing PXR solution, Tween 20 and 5% DMSO (syringe, sample and reference cells)

, After culture cells were harvested by centrifugation and the pellets resuspended in lysis buffer (20 mM Tris pH 7.5, 250 mM NaCl, 5% (v/v) glycerol) supplemented with lysozyme (1 mg ml À 1 ) and a protease inhibitor cocktail tablet (cOmplete, EDTA-free, Roche), and then subjected to sonication. The clarified cell lysate was applied onto a Ni 2 þaffinity column (HisTrap 5 ml; GE Healthcare) equilibrated with lysis buffer supplemented with 10 mM imidazole. The eluted PXR-LBD was then applied onto a gel filtration column, Protein production and purification for structural studies. The human PXR-LBD (130-434) was co-produced with a fragment of the steroid receptor coactivator-1 (SRC-1, 623-710) to enhance PXR stability

, TNC (2.5 molar equivalents), or EE2 þ TNC (2.5 molar equivalents each). Co-crystals with EE2 were obtained in 100 mM NaCl, 100 mM imidazole pH 7.1, 10% (v/v) isopropanol, in 100 mM imidazole pH 7.1, 10% (v/v) isopropanol with TNC alone, and in 50 mM NaCl, 50 mM LiCl, Crystallization. Prior to crystallization assays the purified PXR-LBD (2.4 mg ml À 1 ) was mixed with EE2 (2.5 molar equivalents), vol.100

, Crystals belong to space group P 4 3 2 1 2. The X-ray structures were solved and refined using Phenix (phenix.refine) 43 and COOT 44 . The percentages of residues located in the favoured Ramachandran plot region are 98.1, 98.2 and 97.9% for the PXR À EE2, PXR À TNC and PXR À EE2 À TNC complex structures respectively (calculated with MolProbity 45 ). Data collection and refinement statistics are, Data collection and structure determination. For all complexes, native data were collected from one crystal cryoprotected with 20% (v/v) MPD on the ID23-2 beamline at the European Synchrotron Radiation Facilities (l ¼ 0.8726 Å, 100 K)

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