Amino Acids Important for Ligand Specificity of the Human Constitutive Androstane Receptor^*

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 (Asn¹⁶⁵), 5 (Val¹⁹⁹), 11 (Tyr³²⁶, Ile³³⁰, and Gln³³¹), and 12 (Leu³⁴³ and Ile³⁴⁶) that contribute to the high basal activity of human CAR. Unique residues within helices 3 (Ile¹⁶⁴ and Asn¹⁶⁵), 5 (Cys²⁰² and His²⁰³), and 7 (Phe²³⁴ and Phe²³⁸) were found control the selectivity for CAR activators and inhibitors. A single residue in helix 7 (Phe²⁴³) appears to explain the human/mouse species difference in response of CAR to 17α-ethynyl-3,17β-estradiol.