Differential interaction of 1 alpha,25-dihydroxyvitamin D-3 analogues and their 20-epi homologues with the vitamin D receptor

An important focus of structure-function studies of synthetic ligands for the vitamin D receptor (VDR) concerns the chiral center at carbon 20 of the steroid side chain; 20-epi analogues are 100-10,000 times more potent transcriptionally than the natural hormone 1 alpha,25-dihydroxyvitamin D-3 (1 alpha,2S-(OH)(2)D-3). We have compared the binding properties of three pairs of analogues either with a natural (N) or 20-epi (E) orientation. In intact cells, 45-60% of VDR . N-analogue complexes, but only 5-20% of VDR . E-analogue complexes, dissociated over a 8-h interval, The two groups of ligands induced distinct changes in VDR conformation as revealed by protease clipping assays, Mapping of ligand-VDR binding activity by deletions indicated that amino acids 420-427 were important for high affinity of VDR . N-analogue complexes, but not for VDR . E-analogue complexes, Site-directed mutagenesis revealed that residues 421 and 422 were essential far 1 alpha,25-(OH)(2)D-3-induced conformational changes, high affinity of 1 alpha,25-(OH)(2)D-3 for VDR, and transcriptional activity, but not for binding of its 20-epi analogue, In contrast, deletion of residues 396-427 abolished binding of 1 alpha,25-(OH)(2)D-3, but binding of its 20-epi analogue was still detectable. The results suggest that the ligand-binding domain of VDR has multiple and different contact sites for the two families of side chain-modified ligands, resulting in VDR ligand complexes with different half-lives and transcriptional activities.