Structurally and functionally important amino acids of the agonistic conformation of the human vitamin D receptor

Abstract

The crystal structures of the ligand binding domain of human vitamin D receptor (VDR) complexed with its natural ligand or the superagonists MC1288 or KH1060 have recently been reported. The crystallized ligand binding domain (LBD) of VDR, however, differs from the full-length VDR with respect to deletion of 50 amino acids between its helices 2 and 3. In this study, we investigated structurally and functionally important amino acid interactions within the ligand binding pocket of the full-length VDR in the presence of several synthetic vitamin D(3) analogs. We used site-directed mutagenesis scanning combined with limited proteolytic digestion, electrophoretic mobility shift assay, and reporter gene assay and correlated the findings with the crystal structures of truncated VDR LBD. Our results suggest that structurally different agonists have distinct ligand-receptor interactions and that the amino acid residues H229, D232, E269, F279, and Y295 are critical for the agonistic conformation of the VDR. Our biological data, which were obtained with the full-length VDR, fit well with the crystal structure of the truncated VDR LBD and suggest that removal of the insertion domain between helices 2 and 3 of the receptor does not markedly influence the functionality of the VDR.