Identification of an alternative ligand-binding pocket in the nuclear vitamin D receptor and its functional importance in 1alpha,25(OH)2-vitamin D3 signaling

Abstract

Structural and molecular studies have shown that the vitamin D receptor (VDR) mediates 1alpha,25(OH)2-vitamin D3 gene transactivation. Recent evidence indicates that both VDR and the estrogen receptor are localized to plasma membrane caveolae and are required for initiation of nongenomic (NG) responses. Computer docking of the NG-specific 1alpha,25(OH)2-lumisterol to the VDR resulted in identification of an alternative ligand-binding pocket that partially overlaps the genomic pocket described in the experimentally determined x-ray structure. Data obtained from docking five different vitamin D sterols in the genomic and alternative pockets were used to generate a receptor conformational ensemble model, providing an explanation for how VDR and possibly the estrogen receptor can have genomic and NG functionality. The VDR model is compatible with the following: (i) NG chloride channel agonism and antagonism; (ii) variable ligand-stabilized trypsin digest banding patterns; and (iii) differential transcriptional activity, employing different VDR point mutants and 1alpha,25(OH)2-vitamin D3 analogs.

Fig. 1.

VDR natural and synthetic ligands. (A) The structures of 1,25D, metabolites, and analogs used in this study are shown. The carbon atoms of 1,25D with a hydroxyl moiety (C1, C3, and C25) or a flexible dihedral (360° rotation) are labeled. (Aa and b) Highlighted are the different chemical and physical properties of the two functional halves of the 1,25D molecule (40). (B) Dot maps of 1,25D (27) in a 4.0-kcal energy window were generated by using GMMX (pcmodel V9.0). (B) For side-chain population a, the lowest-energy conformer of the 262 1,25D CD ring fragments is depicted. The C16,17,20,22 dihedral (black arrow) of 98% of these conformers can be grouped into three dihedral population categories defining a window of 20°. For population a, the dihedral range is between +50° and +70° (73%); for population b, between –40° and –60° (14%); and for population c, between +165° and –175° (11%). (C) The α- and β-chair 1,25D A rings are depicted.

Fig. 2.

VDR molecular models depicting the G and A pockets. (A) Ribbon diagrams of the assemblies resulting from molecular modeling of the VDR LBD (x-ray coordinates; Protein Data Bank ID code 1DB1) with 6-s-trans 1,25D in the G pocket (cyan Connolly surface) and JN docked in the A pocket (light-brown Connolly surface). The orange ribbon, H11 and H12, is shown with H12 in the closed, transcriptionally active conformation. The yellow ribbons indicate the orientation of the H2/β-sheet region in VDRwt, whereas the purple ribbons indicate their final position after JN was docked in the A pocket. Important amino acid residues discussed in the text are labeled. (B) Superimposition of the VDR amide backbone atoms of the 1,25D α/β-chair and JN A pocket models. JN's oxygen atoms are red and carbon atoms are green (Colat colors). The α-chair form of 1,25D is orange and the β-chair is pink. The starting orientations of Y295 and H229 are indicated by the thin, cyan wireframe and their final positions are indicated by the thicker, Colat-colored wireframe. (C) Superimposition of the 1,25D/VDR G pocket and the 6-s-cis 1,25D/VDR A pocket (backbone rms = 1.18 Å) models rendered to show the VDR LBD Connolly surface (transparent). The ribbon diagram is color-coded to represent the degree of movement observed when the two models are compared. The red regions indicate >3.0 Å, orange between 2.0 and 3.0 Å, yellow between 1.0 and 2.0 Å, and white <1.0 Å movement when the atomic rms are compared. The location of important, flexible Arg residues discussed in the text are labeled for reference.

Fig. 3.

Effect of 1,25D and its analogs on chloride channel opening in ROS 17/2.8 cells. The chloride currents were elicited by a depolarizing step to 80 mV, in the presence of the indicated sterols. Currents were obtained with glutamate as the permanent anion because seals were more stable and long-lasting than in the presence of Cl^–. Anion currents were isolated from inward Ba⁺² currents after blockage of Ca⁺² channels with 100 μM Cd⁺². The ligand concentrations used were 0.5 nM 1,25D, 0.5 nM 25D, 1.0 nM HL, 1.0 nM HL plus 0.5 nM 1,25D, 1.0 nM JN, and 10 nM E₂ (β-E). The mean effect of each analog was statistically compared with the effect attained by 0.5 nM 1,25D (*, P < 0.05; **, P < 0.01, n = 4–9).

Fig. 4.

Protease sensitivity of ³⁵S-VDRwt in the presence of 1,25D or select analogs. SDS/PAGE gel of partial trypsin digests of ³⁵S-VDR (residues 1–427) incubated with 10^–5 M of the indicated vitamin D sterol. The dense band in the 1,25D lane is the ≈34-kDa fragment (c1) and represents the closed H12 conformer (Fig. 2 A), whereas the dense bands in the 25(OH)D lane are c1 and the ≈30-kDa band (c3). Input represents ³⁵S-VDR not subjected to trypsin treatment.