1. The Crystal Structure of the Nuclear Receptor for Vitamin D Bound to Its Natural Ligand 
N. Rochel, J.M. Wurtz, A. Mitschler, B. Klaholz, D. Moras 
Molecular Cell 
Volume 5, Issue 1, Pages (January 2000)
DOI: /S (00)80413-X

2. Figure 1 Vitamin D Bound to Its Cognate Nuclear Receptor
(a–d) Chemical structure of 1,25(OH)2D3 and analogs. (a) 1,25(OH)2D3: 1α,25-dihydroxyvitamin D3; (c) MC903: 1α,24S-dihydroxy-22-ene-25,26,27-cyclopropylvitamin D3; (d) EB1089: 1α,25-dihydroxy-22,24diene-24,26,27-trihomovitamin D3; (b) KH1060: 1α,25-dihydroxy-20-epi-22-oxa-24,26,27-trihomovitamin D3.
(e) Overall fold of the hVDR ligand-binding domain. The helices are represented as cylinders and β sheets as arrows. The whole structure is colored in gray except the helix H12, shown as a purple cylinder. The ligand is depicted in yellow. The insertion domain location is shown in green. The reconnected residues are indicated, together with their sequence numbering.
Molecular Cell 2000 5, DOI: ( /S (00)80413-X)

3. Figure 2 hVDR Ligand-Binding Domain Functional Assays
(a) Scatchard analysis of 1α,25 (OH)2 vitamin D3 binding by hVDR LBD wild type (open triangle) (118–425) and mutant (closed diamond) (118–425, Δ[165–215]). Scatchard analyses were carried out on dextran/charcoal-derived binding data. The crude extracts of E. coli BL21 (DE3) expressing hVDR wild type or mutant pET 15b were diluted 1000 times and incubated with increasing amounts of 3H-26,27 (Amersham) 1,25(OH)2D3 in 20 mM Tris, 250 mM NaCl, 5 mM dithiothreitol (DTT), 10% glycerol for 16 hr at 4°C. After incubation, 25 μl of dextran/charcoal (1.5%) was added to 25 μl of the protein mixture. After 5 min, the tubes were centrifuged at 13,000 rpm for 5 min. The concentrations of the bound ligand (B) were determined by liquid scintillation counting on supernatant. Total ligand concentrations were measured by liquid scintillation counting on 15 μl of the protein mixture before adding dextran/charcoal. U represents the unbound ligand. Each point represents the average of three values. Data were analyzed by the nonlinear least-square method as described (Claire et al. 1978) for a model of one specific binding site and nonspecific binding sites. The solid and dashed lines represent the fitting of the experimental values for the mutant and wild-type proteins, respectively, with the parameters N = ± nM, Kd = 0.37 ± 0.05 nM, β = ± for the mutant protein and N= 0.10 ± 0.01 nM, Kd = 0.55 ± 0.08 nM, β = ± for the wild-type protein, with N = number of sites, Kd = dissociation constant, and β = nonspecific binding. The experiments have been repeated two times. For the competition assay, the diluted crude extract was incubated with 1 nM of [3H-26,27] 1,25(OH)2D3 and increasing concentrations of 1,25(OH)2D3 analogs for 12–16 hr at 4°C. Bound and free ligand were separated by dextran/charcoal. Nonspecific binding was measured in the presence of 250-fold excess of 1,25(OH)2D3. The experiment has been repeated three times in duplicate.
(b) CAT activities of wild-type and mutant hVDR. The VDR LBDs were fused to the DNA-binding domain of the yeast activator GAL4 (1–147) by cloning the cDNA into the XhoI-BamHI sites of the vector PXJ440 (Xiao et al. 1991). Cos cells were transfected as described (Xiao et al. 1991) with the hVDR wild-type or mutant vector (250 ng) with 2 μg of 17m5-TATA-CAT reporter gene and 2 μg of an internal control recombinant expressing β-galactosidase pCH110lacZ (Pharmacia) completed to 20 mg with carrier DNA. Cells were treated with vehicle (EtOH) or 10–7 M 1,25(OH)2D3. CAT activities, normalized to equal units of β-galactosidase, are expressed relative to the VDR wild type + 1,25(OH)2D3–induced CAT activity (100%). The error has been evaluated as 15%.
Molecular Cell 2000 5, DOI: ( /S (00)80413-X)

4. Figure 3
Close-up Views of Selected Regions of the hVDR-LBD Crystal Structure
(a and b) Comparison of the β sheet region of RARγ (a) and VDR (b) in the same orientation. The RAR and VDR are colored in pink and gray, respectively. Their ligands, retinoic acid and 1,25(OH)2D3, are colored in yellow.
(c) Intramolecular interactions of helix H12 in VDR. The side chains of residue involved in polar stabilization of H12 in its agonist position are shown together with the hydrogen bonds (green dots). The backbone of the protein is colored in gray except for helix H12 in purple. The side chain atoms are colored in gray for carbon, red for oxygen atoms, and blue for nitrogen. The ligand is colored in yellow with the hydroxyl groups shown as red spheres.
(d) Stereo view of 1,25(OH2)D3 in the hVDR ligand-binding pocket. The ligand molecule is shown in the experimental electron density omit map contoured at 1.0 standard deviation. The hydroxyl moieties are depicted as red spheres. Water molecules surrounding the A ring are shown as purple spheres. Hydrogen bonds are shown as green dotted lines. In both pictures, the carbon atoms are colored in gray, and oxygen and nitrogen atoms are colored in red and blue, respectively.
Molecular Cell 2000 5, DOI: ( /S (00)80413-X)