Abstract
The human constitutive androstane receptor (CAR, NR1I3) is an important ligand-activated regulator of oxidative and conjugative enzymes and transport proteins. Because of the lack of a crystal structure of the ligand-binding domain (LBD), wide species differences in ligand specificity and the scarcity of well characterized ligands, the factors that determine CAR ligand specificity are not clear. To address this issue, we developed highly defined homology models of human CAR LBD to identify residues lining the ligand-binding pocket and to perform molecular dynamics simulations with known human CAR modulators. The roles of 22 LBD residues for basal activity, ligand selectivity, and interactions with co-regulators were studied using site-directed mutagenesis, mammalian co-transfection, and yeast two-hybrid assays. These studies identified several amino acids within helices 3 (Asn(165)), 5 (Val(199)), 11 (Tyr(326), Ile(330), and Gln(331)), and 12 (Leu(343) and Ile(346)) that contribute to the high basal activity of human CAR. Unique residues within helices 3 (Ile(164) and Asn(165)), 5 (Cys(202) and His(203)), and 7 (Phe(234) and Phe(238)) were found control the selectivity for CAR activators and inhibitors. A single residue in helix 7 (Phe(243)) appears to explain the human/mouse species difference in response of CAR to 17alpha-ethynyl-3,17beta-estradiol.
Highlights
Nuclear receptors (NRs)1 are ligand-inducible transcription factors that govern many physiological processes
Yeast Two-hybrid Assays—The human CAR and all mutant ligand-binding domain (LBD) were inserted between the EcoRI and BamHI sites in pGBKT7 plasmid (Clontech). pGADT7 plasmids containing human nuclear receptor co-repressor (NCoR) and human steroid receptor coactivator-1 (SRC-1) NR interaction domains (NRID) have been reported [24, 28]
Modulation of Human CAR Activity—Fig. 1A shows that TMPP and clotrimazole enhanced the basal activity of GAL4human CAR LBD by 2.3- and 1.8-fold, whereas EE2 and ANDR inhibited it by 40 and 30%, respectively
Summary
Chemicals—All steroids were at least 99% pure and from Steraloids Inc. (Newport, RI). The normalized luciferase activities are from three to six independent transfections and expressed as mean Ϯ S.E. Yeast Two-hybrid Assays—The human CAR and all mutant LBDs were inserted between the EcoRI and BamHI sites in pGBKT7 plasmid (Clontech). The CAR model without any NRID peptide was equilibrated with decreasing constraints (from 1000 to 100 KJ/mol) on backbone atoms for 400 ps, followed by a free MD simulation of 2.25 ns. For the models with NRID peptides, an equilibration period of 250 ps with constraints of 1000 KJ/mol on the backbone atoms (excluding residues 24 –52 because the VDR template was engineered in this part) was followed by a free MD simulation carried out for 2.25 ns. For dockings of clotrimazole and TMPP in CAR-SRC1 as well as ANDR and EE2 in CAR-NCoR models, MD simulations were performed with the settings described above. Figures were prepared using VMD [53]
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