Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.     

A novel mutation in helix 12 of the vitamin D receptor impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder most often caused by mutations in the vitamin D receptor (VDR). The patient in this study exhibited the typical clinical features of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated serum concentrations of alkaline phosphatase and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D(3)]. The patient did not have alopecia. Assays of the VDR showed a normal high affinity low capacity binding site for [(3)H]1,25-(OH)(2)D(3) in extracts from the patient's fibroblasts. However, the cells were resistant to 1,25-dihydroxyvitamin D action as demonstrated by the failure of the patient's cultured fibroblasts to induce the 24-hydroxylase gene when treated with either high doses of 1,25-(OH)(2)D(3) or vitamin D analogs. A novel point mutation was identified in helix H12 in the ligand-binding domain of the VDR that changed a highly conserved glutamic acid at amino acid 420 to lysine (E420K). The patient was homozygous for the mutation. The E420K mutant receptor recreated by site-directed mutagenesis exhibited many normal properties including ligand binding, heterodimerization with the retinoid X receptor, and binding to vitamin D response elements. However, the mutant VDR was unable to elicit 1,25-(OH)(2)D(3)-dependent transactivation. Subsequent studies demonstrated that the mutant VDR had a marked impairment in binding steroid receptor coactivator 1 (SRC-1) and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex. Taken together, our data indicate that the mutation in helix H12 alters the coactivator binding site preventing coactivator binding and transactivation. In conclusion, we have identified the first case of a naturally occurring mutation in the VDR (E420K) that disrupts coactivator binding to the VDR and causes HVDRR.