Fig. 1. (A) Schematic of the VDR (gray toned olive) and Vitamin D sterol (1,25D structure; see [43] and [44] for review of Vitamin D chemistry) conformational ensembles. All items referred to in the text are labeled in the figure. The mouse-trap model posits that some regions of the VDR are highly flexible in the absence of ligand and that the apo-NR is conformer-1 (1) and that binding of a genomic agonist induces H12 to close (3), thereby activating the transcription factor. The VDR ensemble model posits that ligand can sample the A-pocket regardless of the H12 conformation [11]. (B) Ribbon diagram of the VDR X-ray crystal structure [45]. The C-terminal end of helix-11 (H11) and H12 are colored orange. H2 and the β-sheet are colored yellow and the loops between H1/H2 and H5/β-sheet are colored purple. The position of the β-sheet after JN was successfully docked in the A-pocket is indicated by a gray ribbon. The genomic pocket is defined by a red Connolly surface and labeled 1,25D. The alternative pocket is defined by a blue Connolly surface and labeled JN. The surface area of the His-Rich region (imidizole R-groups rendered in wire-frame) that is exposed to solvent is depicted as a blue dotted surface. All amino acids mentioned in the text are shown in wire-frame and labeled. (C) Approximate 120° clockwise rotation of (B). The histidine residues forming the solvent exposed His-Rich region are labeled. Oxygen atoms of explicit water molecules with X-ray B-values ≤ 30 Å² (average B-value = 24 Å²; pdb code:1DB1) are depicted as red spheres. The coordination of these water molecules is emulated by the red dotted surface. (D) The starting conformer of the β-sheet (line ribbon, colored orange) was superimposed with the final minimized (see methods [32]) VDR LBD conformer (solid gray ribbon) docked with JN (Connolly surface rendered transparent) in the A-pocket. The starting orientation of important A-pocket amino acid residues are displayed in wire-frame, colored orange, and labeled. The final position of these residues are shown in wire-frame and colat colored. The > 3.0 Å movements of Y295 and F150 are depicted by solid red indicators. The positions of oxygen atoms (colored blue), comprising the H₂O-channel that solvates H229, are shown with their Connolly surface rendered transparent. The H229 H-bonding network formed when 1,25D occupies the G-pocket is depicted by red dotted lines, while the H-bonding network when JN occupies the A-pocket is shown by black dotted lines. (E) The figure depicts the H-bonding network (black dotted lines) stabilizing pseudo-6-s-trans 1,25D molecule in the G-pocket with the highlighted ribbon backbone shown in (B) rendered to three lines. The H₂O-channels (blue dotted Connolly surface) that solvate R274 and H397 are rendered so that the oxygen atom that directly coordinates to R274 or H397 is shown as a larger red sphere than the remainder of the channel oxygen atoms.

Fig. 2. Chemical structures of 1α,25(OH)₂-Vitamin D₃ (1,25D), 20-epi-1α,25(OH)₂-Vitamin D₃ (IE), 21-(3′-hydroxy-3′-methylbutyl)-1α,25(OH)₂-D₃ (KH), 1α,25(OH)₂-lumisterol₃ (JN), 23S,25R-1α,25(OH)₂-D₃-26,23-lactone (BS), and 25(OH)-Vitamin D₃ (25D) are shown. Each compound's relative competitive index (RCI) value, and Riverside two letter code, are indicated. The RCI value is obtained using a steroid competition assay (see Methods).

Fig. 3. (A) SDS-PAGE gel of partial trypsin digest of ³⁵S-VDRwt incubated with 10⁻⁵ M 1,25D, BS, and KH or (B) 10⁻⁵ M 1,25D and IE. The three possible H12 conformations that are believed to produce the 34 kDa (c1), 32 kDa (c2), and 30 kDa (c3) VDR-tryptic fragments are shown in Fig. 1A (manuscript submitted).

Calculated G- and A-pocket potential interaction energies (I_E) of 1,25D and five related analogs

The chemical name and structures of 1,25D and its five analogs are presented in Fig. 2.