New insights into Vitamin D sterol-VDR proteolysis, allostery, structure-function from the perspective of a conformational ensemble model.

Recently, we have developed a Vitamin D sterol (VDS)-VDR conformational ensemble model. This model can be broken down into three individual, yet interlinked parts: (a) the conformationally flexible VDS, (b) the apo/holo-VDR helix-12 (H12) conformational ensemble, and (c) the presence of two VDR ligand binding pockets (LBPs); one thermodynamically favored (the genomic pocket, G-pocket) and the other kinetically favored by VDSs (the alternative pocket, A-pocket). One focus of this study is to use directed VDR mutagenesis to (1) demonstrate H12 is stabilized in the transcriptionally active closed conformation (hVDR-c1) by three salt-bridges that span the length of H12 (cationic residues R154, K264 and R402), (2) to elucidate the VDR trypsin sites [R173 (hVDR-c1), K413 (hVDR-c2) and R402 (hVDR-c3)] and (3) demonstrate the apo-VDR H12 equilibrium can be shifted. The other focus of this study is to apply the model to generate a mechanistic understanding to discrepancies observed in structure-function data obtained with a variety of 1alpha,25(OH)(2)-Vitamin D(3) (1,25D) A-ring and side-chain analogs, and side-chain metabolites. We will demonstrate that these structure-function conundrums can be rationalized, for the most part by focusing on alterations in the VDS conformational flexibility and the elementary interaction between the VDS and the VDR A- and G-pockets, relative to the control, 1,25D.

Conservative mutageneic perturbations of amino acids connecting helix 12 in the 1alpha,25(OH)2-D3 receptor (VDR) to the ligand cause significant transactivational effects.

The positioning of helix 12 activation domain of nuclear receptor proteins is critically important for gene regulation. Perturbations of the helix 12 by larger analogs may alter interactions with transcriptional machinery which might give rise to selectivity. To explore the topology of the ligand binding pocket and how the bound ligand conceivably gives rise to altered transcriptional efficiencies, we have targeted 4 hydrophobic residues which contact the 25-carbon of the ligand, 1alpha,25(OH)(2)-vitamin D(3), and made a series of 13 mutants. Substitution of a smaller hydrophobic residue was poorly tolerated compared to a larger one for transactivation. The larger amino acids are likely better tolerated by promoting stronger Van der Waals forces with the ligand. Valine-418 mutants demonstrated an extreme example of this observation with mutation to leucine being transactivationally unaffected with alanine being the most affected of all single mutants. V418L resulted in a 1.3-fold increase in EC(50) for 1,25-D mediated transactivation whereas V418A resulted in a 53-fold increase when compared to wildtype VDR. Importantly, this difference is not explained by ligand binding data but by differential VDR protease sensitivity implying that V418L-VDR mutation assumes a better conformational interaction surface for coactivator than V418A. Importantly, the V418 location may accommodate larger sidechains and may even enhance the interaction with specific nuclear coactivators.

Presence of a truncated form of the vitamin D receptor (VDR) in a strain of VDR-knockout mice.

As part of our studies on the membrane-initiated actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] and its localization in caveolae membrane fractions, we used a vitamin D receptor (VDR)-knockout (KO) mouse model to study the binding of [(3)H]-1alpha,25(OH)(2)D(3) in the presumed absence of the VDR. In this mouse model, known as the Tokyo strain, the second exon of the VDR gene, which encodes the first of the two zinc fingers responsible for DNA binding, was removed, and the resulting animals have been considered to be VDR-null mice. To our surprise, several tissues in these KO mice showed significant (5-50% of that seen in wild-type animals) specific binding of [(3)H]-1alpha,25(OH)(2)D(3) in nuclear and caveolae membrane fractions. The dissociation constants of this binding in samples from VDR-KO and wild-type mice were indistinguishable. RT-PCR analysis of intestinal mRNA from the VDR-KO animals revealed an mRNA that lacks exon 2 but contains exons 3-9 plus two 5'-untranslated exons. Western analysis of intestinal extracts from VDR-KO mice showed a protein of a size consistent with the use of Met52 as the translational start site. Transfection of a plasmid construct containing the sequence encoding the human analog of this truncated form of the receptor, VDR(52-C), into Cos-1 cells showed that this truncated form of the receptor retains full [(3)H]-1alpha,25(OH)(2)D(3) binding ability. This same construct was inactive in transactivation assays using the osteocalcin promoter in CV1 cells. Thus, we have determined that this widely used strain of the VDR-KO mouse can express a form of the VDR that can bind ligand but not activate gene transcription.

A perspective on how the Vitamin D sterol/Vitamin D receptor (VDR) conformational ensemble model can potentially be used to understand the structure-function results of A-ring modified Vitamin D sterols.

The steroid hormone 1alpha,25(OH)(2)-Vitamin D(3) (1,25D) activates both genomic and non-genomic intracellular signaling cascades. It is also well recognized that co-incubation of 1,25D with its C-1 epimer, 1beta,25D (HL), suppresses the efficiency of the non-genomic signal activated by 1,25D alone and that its C-3 epimer, 3alpha-1,25D (HJ) is nearly as potent as 1,25D in suppressing PTH secretion, believed to be propagated by 1,25D's genomic signaling. Both these sterols lack the hypercalcemic effect induced by pharmacological doses of 1,25D and have reduced VDR affinity compared to 1,25D, as measured in a steroid competition assay. Recent functional studies suggest that the VDR is required for both non-genomic and genomic signaling. Along these lines we have recently proposed a Vitamin D sterol/VDR conformational ensemble model that posits the VDR contains two distinct, yet overlapping ligand binding sites, and that the potential differential stabilities of 1,25D and HL in these two pockets can be used to explain their different non-genomic signaling properties. The overlapping region is predominantly occupied by the sterol's A-ring when it is bound to either the genomic ligand binding pocket (G-pocket), defined by X-ray crystallography, or the alternative ligand binding pocket (A-pocket), discovered using in silico techniques (directed docking). Therefore, to gain further insight into the potential application of this model we docked the other A-ring diastereomer [(1beta,3alpha)=HH] of 1,25D and its 1- and 3-deoxy forms (25D and CF, respectively) to the A- and G-pockets to assess their potential stabilities in the pockets, relative to 1,25D. The models were then used to provide putative mechanistic arguments for their known structure-function experimental results. This model may provide new insights into how Vitamin D sterols that uncouple the unwanted hypercalcemic effect from attractive growth inhibitory/differentiation properties can do so by differentially stabilizing different subpopulations of VDR conformational ensemble members.

Applications of the Vitamin D sterol-Vitamin D receptor (VDR) conformational ensemble model.

Over the past 20 years much has been learned about the cellular actions of the steroid hormone 1alpha,25(OH)2-Vitamin D3 (1,25D). Perhaps most importantly structure-function studies led to the discovery that different chemical and physical features of 1,25D are preferred to initiate either exonuclear, non-genomic or endonuclear, genomic cellular signaling. It is well documented that both a 1alpha-OH and 25-OH, and a 6-s-trans, bowl-shaped, sterol conformation are absolutely required for efficient gene transcription, while 6-s-cis locked analogs and 1-deoxy, 25(OH)D3 metabolites activate a variety of non-genomic, rapid responses. These results and the observation that S237 (helix-3; H3) and R274 (H5) are the most static residues in the human 1,25D-Vitamin D receptor (VDR) X-ray construct (see B-values in pdb: 1DB1) and form H-bonds with the 1alpha-OH of 1,25D in the X-ray, genomic pocket (G-pocket), provided the basis for the molecular modeling experiments that led to the discovery of a putative VDR alternative ligand binding pocket (A-pocket). The conformational ensemble model generated from the in silico results provides an explanation for how the VDR can function as a receptor propagating both genomic and non-genomic signaling events. In this report the theoretical gating properties controlling ligand access to the A- and G-pockets will be compared and the model will be used to provide a molecular explanation for the confusing structure-function results pertaining to 1,25D, its side-chain metabolite, 23S,25R-1alpha,25(OH)2-D3-26,23-lactone (BS), and its synthetic two side-chain analog, 21-(3'-hydroxy-3'-methylbutyl)-1alpha,25(OH)2-D3 (KH or Gemini). In addition, evidence that the model is consistent with the pH requirement for Vitamin D sterol-VDR crystallization will be presented.

Effect of 25-hydroxyl group orientation on biological activity and binding to the 1alpha,25-dihydroxy vitamin D3 receptor.

The hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25D), generates many biological actions by interactions with its nuclear receptor (VDR). The presence of a carbon-25 hydroxyl group is necessary for optimizing binding to the VDR. To examine the effect of spatial orientation of the 25-hydroxyl, two pairs of 22,23-allene sidechain analogs were studied. The 22R orientation in analogs HR (52+/-2%) and LA (154+/-19%) resulted in higher affinity binding than the 22S orientation of analogs HQ (21+/-3%) and LB (3.5+/-1.3%; 1,25D=100%). Limited trypsin proteolysis showed that 22R analogs induced VDR conformational changes better able to protect VDR from digestion than 22S analogs. 22R analogs were also able to induce gene transcription at 10-100-fold lower concentrations than 1,25D; 22S analogs were less effective. Analog LA was at least 10-fold more potent than 1,25D at inducing differentiation, while the other analogs were less potent. None of the analogs were as potent as 1,25D in promoting in vivo intestinal calcium absorption or bone calcium mobilization. LA was the most potent of the analogs but required 20-30-fold higher doses than 1,25D. The 25-hydroxyl orientation combined with the 16,17-ene functionality of analog LA enhances its ability to interact with VDR and induce biological actions.

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.

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.

The vitamin D receptor is present in caveolae-enriched plasma membranes and binds 1 alpha,25(OH)2-vitamin D3 in vivo and in vitro.

The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDR(mem)). This study characterized the VDR(mem) present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [(3)H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D K(D) binding dissociation constant = 1-3 nM. Our data collectively support the classical VDR being the VDR(mem) in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [(3)H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r(2) = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [(3)H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [(3)H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

Characterization of five 19-nor-analogs of 1alpha,25(OH)2-Vitamin D3 with 20-cyclopropyl-modified side-chains: implications for ligand binding and calcemic properties.

The steroid hormone 1alpha,25(OH)(2)-Vitamin D(3) [1alpha,25(OH)(2)D(3)] exerts a wide variety of biological actions through one or more receptors/binding proteins. The nuclear Vitamin D receptor (VDR) when bound to its natural ligand, 1alpha,25(OH)(2)D(3), can stimulate transcription of a wide variety of genes. The synthesis of 1alpha,25(OH)(2)D(3) analogs allows the study of structure-function relationships between ligand and the VDR. 1alpha,25(OH)(2)D(3) is a conformationally flexible molecule; specifically the side-chain of the hormone can display a large variety of shapes for its receptor. Here, we describe and analyze the properties of 10 1alpha,25(OH)(2)D(3) analogs modified at the side-chain of which five lack carbon-19 (19-nor) but have a novel 20-cyclopropyl functionality. Analog NG [20,21-methylene-23-yne-26,27-F(6)-19-nor-1alpha,25(OH)(2)D(3)] possesses a respectable binding affinity for the VDR and exhibits a high transcriptional activity (EC(50) approximately 10pM), while retaining low induction of hypercalcemia in vivo in the mouse, making it a primary candidate for further analyses of its anti-proliferative and/or cell differentiating properties.

Evidence that annexin II is not a putative membrane receptor for 1alpha,25(OH)2-vitamin D3.

The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1,25-D(3)) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/p11(2)] was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D(3), based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of AII and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25-D(3) rapid, non-genomic activities, we investigated in vitro the affinity of [(3)H]1,25-D(3) for the AII monomer and AII(2)/p11(2) in the absence and presence of calcium using saturation analysis and gel-filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [(3)H]1,25-D(3) was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII(2)/p11(2). However saturable binding of [(3)H]1,25-D(3) to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K(d) = 3.0 nM; B(max) = 45 fmols/mg total protein). AII was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25-D(3) binding as reported by Baran et al. Our results suggest that AII does not bind 1,25-D(3) in a physiologically relevant manner; however, recent studies linking AII(2)/p11(2) phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down-stream substrate for 1,25-D(3) induced rapid cellular effects.

Novel Gemini analogs of 1alpha,25-dihydroxyvitamin D(3) with enhanced transcriptional activity.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), exerts its effects through regulation of target gene transcription. Configuration at C-20 of 1,25(OH)(2)D(3) is important in determining potency, as shown by the high potency of analogs with inverted configuration at C-20 (20-epi compounds). Gemini analogs of 1,25(OH)(2)D(3) contain two side chains, combining a C-20-normal with a C20-epi side chain. We studied the potency of analogs combining double (Gemini) side chains with a 23-triple bond and a C-26,27-hexafluoro substitution in either the 20-epi (analog 20R) or 20-normal (analog 20S) side chain. These novel Gemini analogs were 8-50-fold more potent than 1,25(OH)(2)D(3) in inducing U937, HL-60G, and THP-1 differentiation and 5-50-fold more potent in inducing transcription from the osteocalcin vitamin D response element or the 25-hydroxyvitamin D(3)-24-hydroxylase (24OHase) promoter. In vivo, following i.p. injection in vitamin D-deficient mice, the 20S analog induced significantly higher levels of calbindin-D(9K) mRNA in intestine, and 24OHase and calbindin-D(28K) in kidney than 1,25(OH)(2)D(3) or analog 20R. Increased potency did not correlate with ligand-receptor binding affinity. In GST-pull down assays using in vitro translated VDR, Gemini analogs showed equivalent (or even attenuated) potency to 1,25(OH)(2)D(3) in recruiting cofactors DRIP205 and GRIP-1 to VDR. However, Gemini analogs were up to 15-fold more potent than 1,25(OH)(2)D(3) in recruiting the same cofactors to VDR in GST-pull down assays using equal amounts of VDR from nuclear extracts of VDR transfected and hormone treated (24 hr) COS-7 cells. Deletion of C-19 in either 20S or 20R Gemini analogs resulted overall in slightly less potent analogs compared to Gemini itself. We conclude that enhanced potency of the novel Gemini analogs is at least partly due to increased metabolic stability of the analogs, resulting in more cofactor binding and elevated levels of transcription.

A specific binding protein/receptor for 1alpha,25-dihydroxyvitamin D(3) is present in an intestinal caveolae membrane fraction.

The steroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] produces biological responses by interaction with both a well-characterized nuclear receptor (VDR(nuc)) to regulate gene transcription and with an as-yet uncharacterized membrane-associated protein/receptor (VDR(mem)) to generate a variety of rapid, non-genotropic responses. We report for the first time that [3H]1alpha,25(OH)(2)D(3) binds with high affinity to a chick duodenal caveolae-enriched membrane fraction (CMF) isolated without the use of detergents. Caveolae are plasma membrane invaginations implicated in signal transduction and molecular transport processes. Using the CMF fraction as a possible source of VDR(mem), we found that the in vitro binding of [3H]1alpha,25(OH)(2)D(3) was ligand dependent and saturable; the K(D) and B(max) were 1.3+/-0.6nM and 29+/-11fmol 1,25(OH)(2)D(3)/mg protein (n=17), respectively. Immunoblot analysis of the CMF confirms the presence of caveolin-1, a marker protein for membranes with caveolae. Therefore, chick CMF may represent a good source for isolation and characterization of the putative VDR(mem) for 1alpha,25(OH)(2)D(3).

Update on biological actions of 1alpha,25(OH)2-vitamin D3 (rapid effects) and 24R,25(OH)2-vitamin D3.

All biologic responses to vitamin D are now known to arise as a consequence of the metabolism of this seco-steroid into its two principal biologically active metabolites 1alpha,25(OH)(2)-vitamin D(3) (1ALPHA;,25(OH)(2)D(3)) and 24R,25(OH)(2)-vitamin D(3) (24R,25(OH)(2)D(3)). 1alpha,25(OH)(2)D(3) is the dominant metabolite and produces a wide array of biological responses via interacting both with the classical vitamin D nuclear receptor (VDR(nuc)) that regulates gene transcription in over 30 target organs and with a putative cell membrane receptor (VDR(mem1,25)) that mediates rapid (within seconds to minutes) biological responses. Ligand occupancy of VDR(mem1,25) is linked to signal transduction systems that can mediate the opening of Ca(2+) and chloride voltage gated channels as well as activation of MAP-kinase. MAP-kinase activation in some cells containing VDR(mem1,25)+VDR(nuc) then results in "cross-talk" from VDR(mem1,25) to VDR(nuc) which modulates transactivation of 1alpha,25(OH)(2)D(3) responsive gene promoters. The 24R,25(OH)(2)D(3) metabolite has been shown to be an essential hormone for the process of bone fracture healing. The activity of the enzyme responsible for the production of 24R,25(OH)(2)D(3), the renal 25(OH)D-24-hydroxylase, becomes elevated within 4-11 days after imposition of a tibial fracture, thereby increasing the blood concentrations of 24R,25(OH)(2)D(3) by threefold. The 24R,25(OH)(2)D(3) likely initiates its biological responses via binding to the ligand binding domain of a second cell membrane receptor, the VDR(mem24,25), which is stereospecific for 24R,25(OH)(2)D(3) in comparison with 24S,25(OH)(2)D(3) and 1alpha,25(OH)(2)D(3). This report summarizes the status of several current research frontiers in this arena of the vitamin D endocrine system.

Molecular tools for study of genomic and rapid signal transduction responses initiated by 1 alpha,25(OH)(2)-vitamin D(3).

The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) [1 alpha,25(OH)(2)D(3)] mediates through its widely distributed nuclear receptor (VDR(nuc)) regulation of gene transcription (genomic responses) and through a putative membrane receptor (VDR(mem)) a variety of rapid responses. Rapid responses studied in our laboratories include opening of voltage-gated calcium and chloride channels in ROS 17/2.8 osteoblast cells, activation of MAP-kinase in human leukemia NB4 cells and chick intestinal cells, release of insulin by rat pancreatic beta-cells, and in chick duodena transcaltachia (the rapid hormonal stimulation of intestinal Ca(2+) transport). 1 alpha,25(OH)(2)D(3) is conformationally flexible (side chain, seco B-ring and A-ring) and accordingly is able to generate a large array of different shapes to serve as ligands for available receptors (VDR(nuc) and VDR(mem)) in the vitamin D endocrine system. Our laboratories have utilized a number of conformationally restricted analogs of 1 alpha,25(OH)(2)D(3) (from a library of several hundred analogs) to evaluate the preferred shape of the ligands for rapid and genomic responses. The determination of the X-ray structure of the 1 alpha,25(OH)(2)D(3)-occupied VDR(nuc) revealed that the preferred ligand shape was a twisted 6-s-trans bowl shape [Molecular Cell 5 (2000) 173-179]. Optimal agonists for genomic responses include 1 alpha,25(OH)(2)D(3) and other side chain conformationally flexible analogs such as 20-epi-1 alpha,25(OH)(2)D(3) [approximately equal to 200-500-fold more potent than 1 alpha,25(OH)(2)D(3)] and 21-(3'-hydroxy-3-methylbutyl)-1 alpha,25(OH)(2)D(3) [an analog with two side chains] all which can achieve the preferred VDR(nuc) shape. In contrast, rapid responses require a 6-s-cis shape of the agonist ligand such as can be achieved by the natural hormone 1 alpha,25(OH)(2)D(3) or by analogs permanently locked in the 6-s-cis shape such as 1 alpha,25(OH)(2)lumisterol(3) or 1 alpha,25(OH)(2)-7-dehydrocholesterol. Additionally, we have discovered analogs that are specific in their antagonist properties for either rapid or genomic responses. Thus, 1 beta,25(OH)(2)D(3) is an antagonist of only rapid responses [via the VDR(mem)], while 23S-25-dehydro-1 alpha,25(OH)D(3)-26,23-lactone is an antagonist of only nuclear responses [via the VDR(nuc)]. In conclusion, we have presented evidence that 1 alpha,25(OH)(2)D(3) mediated rapid response and genomic response signal transduction pathways utilize differing shapes of ligand, both as agonists and antagonists.