ABSTRACT: The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1?,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 ?M and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1?,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.
