The role of residue C410 on activation of the human vitamin D receptor by various ligands.

Nuclear receptors (NRs) are ligand-activated transcription factors that regulate the expression of genes involved in biologically important processes. The human vitamin D receptor (hVDR) is a member of the NR superfamily and is responsible for maintaining calcium and phosphate homeostasis. This receptor is activated by its natural ligand, 1α, 25-dihydroxyvitamin D(3) (1α, 25(OH)(2)D(3)), as well as bile acids such as lithocholic acid (LCA). Disruption of molecular interactions between the hVDR and its natural ligand result in adverse diseases, such as rickets, making this receptor a good target for drug discovery. Previous mutational analyses of the hVDR have mainly focused on residues lining the receptor's ligand binding pocket (LBP) and techniques such as alanine scanning mutagenesis and site-directed mutagenesis. In this work, a rationally designed hVDR library using randomized codons at selected positions provides insight into the role of residue C410, particularly on activation of the receptor by various ligands. A variant, C410Y, was engineered to bind LCA with increased sensitivity (EC(50) value of 3 μM and a 34-fold activation) in mammalian cell culture assays. Furthermore, this variant displayed activation with a novel small molecule, cholecalciferol (chole) which does not activate the wild-type receptor, with an EC(50) value of 4 μM and a 25-fold activation. The presence of a bulky residue at this position, such as a tyrosine or phenylalanine, may contribute towards molecular interactions that allow for the enhanced activation with LCA and novel activation with chole. Additional bulk at the same end of the pocket, such as in the case of the variant H305F; C410Y enhances the receptor's sensitivity for these ligands further, perhaps due to the filling of a cavity. The effects of residue C410 on specificity and activation with the different ligands studied were unforeseen, as this residue does not line the hVDR's LBP. Further investigating of the structure-function relationships between the hVDR and its ligands, including the mutational tolerance of residues within as well as outside the LBP, is needed for a comprehensive understanding of the functionality and interactions of the receptor with these ligands and for development of new small molecules as potential therapeutic drugs.