Vitamin D Receptor Ligand-binding Domain Research Articles

RF15 | PMON293 Metabolic activation of tachysterol to biologically active hydroxyderivatives that act on VDR, AhR, LXRs and PPARγ receptors

Abstract Absorption of ultraviolet B (UVB) radiation by the B ring of 7-dehydrocholesterol (7-DHC) leads to the transformation of 7-DHC to previtamin D3, which after absorption of additional UVB isomerizes to tachysterol3 (T3) and lumisterol3 (L3). Previously we demonstrated that CYP11A1 can hydroxylate the side chain of vitamin D3 (D3), 7-DHC and L3. Similarly CYP27A1 can hydroxylate the side chain of L3 to biologically active hydroxyderivatives. In a continuation of these studies, we report that CYP11A1 and CYP27A1 hydroxylate T3 to 20S(OH)T3 and 25(OH)T3, respectively, plus minor unidentified hydroxyderivatives. Both 20S(OH)T3 and 25(OH)T3were detected in the human epidermis and serum. T3 was also detected in human serum and was present at a concentration of 7.3±2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 . They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR-coupled GFP (VDR-GFP) from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay revealed marked activation of AhR by 20S(OH)T3, a smaller effect by 25(OH)T3 and only minimal activation by T3. The T3 hydroxyderivatives showed high affinity binding to the ligand binding domain (LBD) of the liver X receptor (LXR) α and β, and to the peroxisome proliferator-activated receptor γ (PPARg) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3, comparable to their previously characterized ligands. The scores for binding to the non-genomic site of the VDR were very low indicating a lack of interaction with hydroxy-T3 ligands. In conclusion, we have identified 20S(OH)T3 and 25(OH)T3 in the human epidermis and serum, identified CYP enzymes responsible for their production, demonstrated phenotypic effects on skin cells, and identified VDR, AhR, LXRs and PPARγ, and possibly RORs, as their genomic receptor targets. Thus CYP11A1, aside from its role in steroidogenesis, not only metabolizes vitamin D3, 7-DHC and L3 to biologically active metabolites, but also activates T3 to biologically active 20S(OH)T3. Similarly, CYP27A1, previously reported to act on L3, D3 and 7DHC, also activates T3 via hydroxylation at C25. We believe that these novel findings open new areas for future studies on the role of active forms of T3, not only in the skin, but also in systemic physiology and pathology. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Advances in Vitamin D Receptor Function and Evolution Based on the 3D Structure of the Lamprey Ligand-Binding Domain.

1α,25-dihydroxyvitamin D3 (1,25D3) regulates many physiological processes in vertebrates by binding to the vitamin D receptor (VDR). Phylogenetic analysis indicates that jawless fishes are the most basal vertebrates exhibiting a VDR gene. To elucidate the mechanism driving VDR activation during evolution, we determined the crystal structure of the VDR ligand-binding domain (LBD) complex from the basal vertebratePetromyzon marinus, sea lamprey (lVDR). Comparison of three-dimensional crystal structures of the lVDR-1,25D3 complex with higher vertebrate VDR-1,25D3 structures suggests that 1,25D3 binds to lVDR similarly to human VDR, but with unique features for lVDR around linker regions between H11 and H12 and between H9 and H10. These structural differences may contribute to the marked species differences in transcriptional responses. Furthermore, residue co-evolution analysis of VDR across vertebrates identifies amino acid positions in H9 and the large insertion domain VDR LBD specific as correlated.

Mechanism of Vitamin D Receptor Ligand-Binding Domain Regulation Studied by gREST Simulations.

The vitamin D receptor ligand-binding domain (VDR-LBD) undergoes conformational changes upon ligand binding. In this nuclear receptor family, agonistic or antagonistic activities are controlled by the conformation of the helix (H)12. However, all crystal structures of VDR-LBD reported to date correspond to the active H12 conformation, regardless of agonist/antagonist binding. To understand the mechanism of VDR-LBD regulation structurally, conformational samplings of agonist- and antagonist-bound rat VDR-LBD were performed using the generalized replica exchange with solute tempering (gREST) method. The gREST simulations demonstrated different structural responses of rat VDR-LBD to agonist or antagonist binding, whereas in conventional molecular dynamics simulations, the conformation was the same as that of the crystal structures, regardless of agonist/antagonist binding. In the gREST simulations, a spontaneous conformational change of H12 was observed only for the antagonist complex. The different responses to agonist/antagonist binding were attributed to hydrophobic core formation at the ligand-binding pocket and cooperative rearrangements of H11. The gREST method can be applied to the examination of structure-activity relationships for multiple VDR-LBD ligands.

Structural Analysis of VDR Complex with ZK168281 Antagonist.

Vitamin D receptor (VDR) antagonists prevent the VDR activation function helix 12 from folding into its active conformation, thus affecting coactivator recruitment and antagonizing the transcriptional regulation induced by 1α,25-dihydroxyvitamin D3. Here, we report the crystal structure of the zebrafish VDR ligand-binding domain in complex with the ZK168281 antagonist, revealing that the ligand prevents optimal folding of the C-terminal region of VDR. This interference was confirmed by hydrogen-deuterium exchange mass spectrometry (HDX-MS) in solution.

Development of novel lithocholic acid derivatives as vitamin D receptor agonists

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells. Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6 bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated derivatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis of the complex of VDR ligand-binding domain (LBD) with 6.

Vitamin D and Its Synthetic Analogs.

For many individuals, in particular during winter, supplementation with the secosteroid vitamin D3 is essential for the prevention of bone disorders, muscle weakness, autoimmune diseases, and possibly also different types of cancer. Vitamin D3 acts via its metabolite 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as potent agonist of the transcription factor vitamin D receptor (VDR). Thus, vitamin D directly affects chromatin structure and gene regulation at thousands of genomic loci, i.e., the epigenome and transcriptome of its target tissues. Modifications of 1,25(OH)2D3 at its side-chain, A-ring, triene system, or C-ring, alone and in combination, as well as nonsteroidal mimics provided numerous potent VDR agonists and some antagonists. The nearly 150 crystal structures of VDR’s ligand-binding domain with various vitamin D compounds allow a detailed molecular understanding of their action. This review discusses the most important vitamin D analogs presented during the past 10 years and molecular insight derived from new structural information on the VDR protein.

Novel split luciferase-based biosensors for evaluation of vitamin D receptor ligands and their application to estimate CYP27B1 activity in living cells

Recently, we successfully generated a novel detection system for vitamin D receptor (VDR) ligands in vivo and in vitro, using a split-luciferase technique called the LucN-LBD-LucC biosensor that is a chimeric fusion protein of firefly luciferase with the ligand binding domain (LBD) of VDR. In this system, the luciferase light intensity of the LucN-LBD-LucC biosensor was decreased by binding of VDR ligands. Although this system is quite useful for evaluation of VDR ligands in a short time, the sensitivity of the LucN-LBD-LucC biosensor is not high enough. In this study, LXXLL motif peptides involved in the interaction between LBD and coactivators, such as the steroid receptor coactivator-1 (SRC-1), transcriptional intermediary factor 2 (TIF2), and the vitamin D receptor interacting protein 205 (DRIP205) were each inserted between LucN and LBD of the LucN-LBD-LucC biosensor. Surprisingly, the resulting LucN-LXXLL-LBD-LucC biosensor increased the light intensity in response to natural VDR ligands. This high-sensitivity biosensor system may be a powerful tool for discovery of high-affinity ligands for the mutant VDR. In addition, we have successfully estimated the activity of the wild-type and mutant CYP27B1 using the LucN-LXXLL-LBD-LucC biosensor in living cells within 90 min.

Novel screening system for high-affinity ligand of heredity vitamin D-resistant rickets-associated vitamin D receptor mutant R274L using bioluminescent sensor

Hereditary vitamin D-resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR) gene. Arg274 located in the ligand binding domain (LBD) of VDR is responsible for anchoring 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by forming a hydrogen bond with the 1α-hydroxyl group of 1α,25(OH)2D3. The Arg274Leu (R274L) mutation identified in patients with HVDRR causes a 1000-fold decrease in the affinity for 1α,25(OH)2D3, and dramatically reduces vitamin D- related gene expression. Recently, we successfully constructed fusion proteins consisting of split-luciferase and LBD of the VDR. The chimeric protein LucC-LBD-LucN, which displays the C-terminal domain of luciferase (LucC) at its N-terminus, can detect and discriminate between VDR agonists and antagonists. The LucC-LBD (R274L)-LucN was constructed to screen high-affinity ligands for the mutant VDR (R274L). Of the 33 vitamin D analogs, 5 showed much higher affinities for the mutant VDR (R274L) than 1α,25(OH)2D3, and 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-(OH)2D3 showed the highest affinity. These compounds might be potential therapeutics for HVDRR caused by the mutant VDR (R274L).

The conformational dynamics of H2-H3n and S2-H6 in gating ligand entry into the buried binding cavity of vitamin D receptor.

Crystal structures of holo vitamin D receptor (VDR) revealed a canonical conformation in which the ligand is entrapped in a hydrophobic cavity buried in the ligand-binding domain (LBD). The mousetrap model postulates that helix 12 is positioned away from the domain to expose the interior cavity. However, the extended form of helix 12 is likely due to artifacts during crystallization. In this study, we set out to investigate conformational dynamics of apo VDR using molecular dynamics simulation on microsecond timescale. Here we show the neighboring backbones of helix 2-helix 3n and beta strand 2-helix 6 of LBD, instead of the helix 12, undergo large-scale motion, possibly gating the entrance of ligand to the ligand binding domain. Docking analysis to the simulated open structure of VDR with the estimated free energy of −37.0 kJ/mol, would emphasise the role of H2-H3n and S2-H6 in facilitating the entrance of calcitriol to the LBD of VDR.

Relationship between Structure and Conformational Change of the Vitamin D Receptor Ligand Binding Domain in 1α,25-Dihydroxyvitamin D3 Signaling.

Vitamin D Receptor (VDR) belongs to the nuclear receptor (NR) superfamily. Whereas the structure of the ligand binding domain (LBD) of VDR has been determined in great detail, the role of its amino acid residues in stabilizing the structure and ligand triggering conformational change is still under debate. There are 13 α-helices and one β-sheet in the VDR LBD and they form a three-layer sandwich structure stabilized by 10 residues. Thirty-six amino acid residues line the ligand binding pocket (LBP) and six of these residues have hydrogen-bonds linking with the ligand. In 1α,25-dihydroxyvitamin D3 signaling, H3 and H12 play an important role in the course of conformational change resulting in the provision of interfaces for dimerization, coactivator (CoA), corepressor (CoR), and hTAFII 28. In this paper we provide a detailed description of the amino acid residues stabilizing the structure and taking part in conformational change of VDR LBD according to functional domains.

Fine tuning of agonistic/antagonistic activity for vitamin D receptor by 22-alkyl chain length of ligands: 22S-Hexyl compound unexpectedly restored agonistic activity

1α,25-Dihydroxyvitamin D3 exerts its actions by binding to vitamin D receptor (VDR). We are continuing the study related to the alteration of pocket structure of VDR by 22-alkyl substituent of ligands and the relationships between the alteration and agonistic/antagonistic activity. Previously we reported that compounds 2 (22-H), 3 (22S-Et), and 4 (22S-Bu) are VDR agonist, partial agonist and antagonist, respectively. Here, we describe the synthesis and biological evaluation of 22S-hexyl analog 5 (22S-Hex), which was designed to be a stronger VDR antagonist than 4. Unexpectedly, 5 showed partial agonistic but not antagonistic activity when bound to VDR, indicating that it is not necessarily true that the bulkier the side chain is, the stronger the antagonistic activity will be. X-ray crystallographic analysis of the VDR–ligand-binding domain (VDR–LBD) accommodating compound 5 indicated that the partial agonist activity of 5 is dependent on the mixed population of the agonistic and antagonistic conformations. Binding of compound 5 may not bring the complex into the only antagonistic conformation due to the large conformational change of the VDR–LBD. From this study it was found that fine tuning of agonistic/antagonistic activity for VDR is possible by 22-alkyl chain length of ligands.

Development of Novel Bioluminescent Sensor to Detect and Discriminate between Vitamin D Receptor Agonists and Antagonists in Living Cells.

Active forms of vitamin D regulate the expression of multiple genes that play essential roles in calcium and phosphate homeostasis, cell differentiation, and the immune system via the vitamin D receptor (VDR). Many vitamin D analogs have been synthesized for clinical use in the treatment of type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia, and breast cancer. We have constructed two fusion proteins containing split-luciferase and the ligand binding domain (LBD) of the VDR designated as LucN-LBD-LucC and LucC-LBD-LucN. Remarkably, the LucC-LBD-LucN, which has the C-terminal domain of luciferase at the N-terminus of the fusion protein, was a significantly better biosensor than LucN-LBD-LucC. Addition of the VDR agonists to COS-7 cells expressing LucC-LBD-LucN dramatically reduced luciferase activity. In contrast, the VDR antagonist significantly increased the chimeric luciferase activity in a dose- and time-dependent manner. Our results on chimeric luciferases containing the LBDs of mutant VDRs derived from patients with vitamin D-dependent type II rickets indicated that our system could detect a conformational change of the LBD of the VDR likely based on a positional change of the helix 12, which occurs upon ligand binding. This novel system to detect and discriminate between VDR agonists and antagonists could be useful for the screening and identification of chemical compounds that bind to normal or mutant VDRs with high affinity.

A Vitamin D Receptor Selectively Activated by Gemini Analogs Reveals Ligand Dependent and Independent Effects

The bioactive form of vitamin D [1,25(OH)2D3] regulates mineral and bone homeostasis and exerts potent anti-inflammatory and antiproliferative properties through binding to the vitamin D receptor (VDR). The 3D structures of the VDR ligand-binding domain with 1,25(OH)2D3 or gemini analogs unveiled the molecular mechanism underlying ligand recognition. On the basis of structure-function correlations, we generated a point-mutated VDR (VDR(gem)) that is unresponsive to 1,25(OH)2D3, but the activity of which is efficiently induced by the gemini ligands. Moreover, we show that many VDR target genes are repressed by unliganded VDR(gem) and that mineral ion and bone homeostasis are more impaired in VDR(gem) mice than in VDR null mice, demonstrating that mutations abolishing VDR ligand binding result in more severe skeletal defects than VDR null mutations. As gemini ligands induce VDR(gem) transcriptional activity in mice and normalize their serum calcium levels, VDR(gem) is a powerful tool to further unravel both liganded and unliganded VDR signaling.

HDX reveals unique fragment ligands for the vitamin D receptor

Modulation of the vitamin D receptor (VDR) with a ligand has the potential to be useful for the oral treatment of osteoporosis. One component of our lead generation strategy to identify synthetic ligands for VDR included a fragment based drug design approach. Screening of ligands in a VDR fluorescence polarization assay and a RXR/VDR conformation sensing assay resulted in the identification of multiple fragment hits (lean >0.30). These fragment scaffolds were subsequently evaluated for interaction with the VDR ligand binding domain using hydrogen–deuterium exchange (HDX) mass spectrometry. Significant protection of H/D exchange was observed for some fragments in helixes 3, 7, and 8 of the ligand binding domain, regions which are similar to those seen for the natural hormone VD3. The fragments appear to mimic the A-ring of VD3 thereby providing viable starting points for synthetic expansion.

Structural insights into the molecular mechanism of vitamin D receptor activation by lithocholic acid involving a new mode of ligand recognition.

The vitamin D receptor (VDR), an endocrine nuclear receptor for 1α,25-dihydroxyvitamin D3, acts also as a bile acid sensor by binding lithocholic acid (LCA). The crystal structure of the zebrafish VDR ligand binding domain in complex with LCA and the SRC-2 coactivator peptide reveals the binding of two LCA molecules by VDR. One LCA binds to the canonical ligand-binding pocket, and the second one, which is not fully buried, is anchored to a site located on the VDR surface. Despite the low affinity of the alternative site, the binding of the second molecule promotes stabilization of the active receptor conformation. Biological activity assays, structural analysis, and molecular dynamics simulations indicate that the recognition of two ligand molecules is crucial for VDR agonism by LCA. The unique binding mode of LCA provides clues for the development of new chemical compounds that target alternative binding sites for therapeutic applications.

A mixed population of antagonist and agonist binding conformers in a single crystal explains partial agonism against vitamin D receptor: active vitamin D analogues with 22R-alkyl group.

We are continuing to study the structural basis of vitamin D receptor (VDR) agonism and antagonism by using 22S-alkyl vitamin D analogues. Here we report the synthesis and biological evaluation of 22R-alkyl analogues and the X-ray crystallographic analysis of vitamin D receptor ligand-binding domain (VDR-LBD) complexed with a 22R-analogue. VDR-LBD complexed with the partial agonist 8a showed that 8a binds to VDR-LBD with two conformations, one of which is the antagonist/VDR-LBD complex structure and the other is the agonist/VDR-LBD complex structure. The results indicate that the partial agonist activity of 8a depends on the sum of antagonistic and agonistic activities caused by the antagonist and agonist binding conformers, respectively. The structural basis observed here must be applicable to the partial agonism of other ligand-dependent nuclear receptors. This is the first report describing the trapping of a conformational subset of the ligand and the nuclear receptor in a single crystal.

Enteral calcium infusion used successfully as treatment for a patient with hereditary vitamin D resistant rickets (HVDRR) without alopecia: A novel mutation

BackgroundWe report a novel mutation in a case of hereditary vitamin D resistant rickets (HVDRR) without alopecia and successful management of this condition with the intravenous formulation of calcium chloride delivered via gastric tube. Clinical CaseA 22month old male (length −3.4 SDS; weight −2.1 SDS) presented with failure to thrive, short stature, severe hypocalcemia and gross motor delay. He did not have alopecia. Initial blood tests and history were thought possibly to suggest vitamin D deficiency rickets: calcium 5.1mg/dL, (8.8–10.8); phosphorus 4.1mg/dL, (4.5–5.5); alkaline phosphatase 1481 U/L (80–220); intact PTH 537.1pg/mL (10–71). Subsequently, vitamin D studies returned that were consistent with HVDRR: 25-hydroxyvitamin D 34ng/mL (20–100); 1,25-dihydroxyvitamin D 507pg/mL. This diagnosis was confirmed by DNA sequencing. His subsequent clinical course was complicated by the fact that IV calcium was not a viable option for this patient, and his calcium levels could not be well controlled on oral calcium citrate or calcium glubionate therapy. Eventually, we were able to maintain calcium levels above 8mg/dL using the intravenous preparation of calcium chloride administered via gastric tube. Genetic StudiesA unique homozygous T to C base substitution was found in exon 6 in the vitamin D receptor (VDR) gene. This mutation causes leucine to be converted to proline at position 227 in helix 3 in the VDR ligand binding domain (LBD). The mutation rendered the VDR non-functional, leading to HVDRR, with absence of alopecia. ConclusionHVDRR should be considered in a patient with profound hypocalcemia which is refractory to conventional therapy of vitamin D deficiency rickets even without evidence of alopecia. We report the first case of HVDRR with a novel mutation in the LBD that was successfully treated with enteral treatment using a calcium chloride infusion.

Synthetically Accessible Non-Secosteroidal Hybrid Molecules Combining Vitamin D Receptor Agonism and Histone Deacetylase Inhibition

1,25-Dihydroxyvitamin D(3) (1,25D), the hormonal form of vitamin D, and several analogs have failed as monotherapies for cancer because of poor efficacy or acquired resistance. However, 1,25D analogs are amenable to bifunctionalization. Preclinical studies have revealed combinatorial effects of 1,25D analogs and histone deacetylase inhibitors (HDACi). Secosteroidal hybrid molecules combining vitamin D receptor (VDR) agonism with HDACi displayed enhanced efficacy but are laborious to synthesize. Here, we have developed easily assembled, fully integrated, non-secosteroidal VDR agonist/HDACi hybrids. The most promising are full VDR agonists with ~10-fold lower potency than 1,25D. Structure/function studies revealed that antiproliferative activity against 1,25D-resistant squamous carcinoma cells required VDR agonism and HDACi. Remarkably, modeling and X-ray crystallography reveal non-secosteroidal hybrids bind in the VDR ligand binding domain in the opposite orientation of their secosteroidal counterparts.

DDX5 is a multifunctional co-activator of steroid hormone receptors

The vitamin D receptor (VDR), an evolutionarily conserved member of the nuclear receptor superfamily, links the metabolically activated vitamin D ligand, calcitriol, with its vitamin D-responsive target genes that are implicated in diverse physiological processes. By genome-wide protein–protein interaction screening of a keratinocyte cDNA library using VDR as bait, we found that the DEAD box RNA helicase p68, also referred to as DDX5, directly interacts with VDR. Domain analysis reveals that the ligand-binding domain of VDR is responsible for the binding, an interaction typical of NR co-activators. Interestingly, the VDR interacting domain of DDX5 lacks a LXXLL-motif and interaction analysis of helix 12 VDR mutants E420K, E420Q and L417S, known to decrease binding affinity of LxxLL motif-containing co-activators showed no change in their interactions. As further support that this novel interactor might be involved in vitamin D-stimulated transcriptional regulation, we demonstrate that VDR and DDX5 co-localize within the nuclei of HaCaT keratinocytes and sub-cellular protein fractions. In vivo validation studies demonstrate, that overexpression of DDX5 has the capability to enhance both, calcitriol-dependent transcription of known response genes and an extrachromosomal DR3-type reporter response. In agreement with this, shRNA based knock-down of DDX5 in keratinocytes compensates for this particular response. Finally, our findings reveal parallels between the VDR-DDX5 interaction and the well-characterized interaction between DDX5 and human estrogen receptor α and the androgen receptor, thus underscoring the physiological significance of the novel protein–protein interaction.

Ligand-Specific Structural Changes in the Vitamin D Receptor in Solution

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)₂D₃], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D₃ [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D₃ [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators.