Ligand-mediated conformational changes of the VDR are required for gene transactivation

The central element of the molecular switch of nuclear 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) signaling is the ligand-binding domain (LBD) of the Vitamin D receptor (VDR), which can be stabilized by 1alpha,25(OH)(2)D(3) or its analogues in to agonistic, antagonistic or inverse agonistic conformations. The positioning of helix 12 of the LBD is of most critical importance for these conformations, because it determines the distance between the charge clamp amino acids K246 and E420 that are essential for VDR-coactivator (CoA) interaction. Most VDR ligands have been identified as agonists and only a few (e.g., ZK168281 and TEI-9647) as pure or partial antagonists. Antagonists induce corepressor (CoR) dissociation from the VDR but prevent completely or partially CoA interaction and thus transactivation. Gemini is a 1alpha,25(OH)(2)D(3) analogue with two identical side chains that despite its significantly increased volume binds to the VDR and acts under most conditions as an agonist. Interestingly, supramolar CoR concentrations shift Gemini from an agonist to an inverse agonist, which actively recruits CoR to the VDR and thus mediates repression of 1alpha,25(OH)(2)D(3) target genes. Gemini is the first described (conditional) inverse agonist to an endocrine nuclear receptor (NR) and may function as a sensor for cell-specific CoA/CoR ratios.     

Antagonist- and inverse agonist-driven interactions of the vitamin D receptor and the constitutive androstane receptor with corepressor protein

Ligand-dependent signal transduction by nuclear receptors (NRs) includes dynamic exchanges of coactivator (CoA) and corepressor (CoR) proteins. Here we focused on the structural determinants of the antagonist- and inverse agonist-enhanced interaction of the endocrine NR vitamin D receptor (VDR) and the adopted orphan NR constitutive androstane receptor (CAR) from two species with the CoR NR corepressor. We found that the pure VDR antagonist ZK168281 and the human CAR inverse agonist clotrimazole are both effective inhibitors of the CoA interaction of their respective receptors, whereas ZK168281 resembled more the mouse CAR inverse agonist androstanol in its ability to recruit CoR proteins. Molecular dynamics simulations resulted in comparable models for the CoR receptor interaction domain peptide bound to VDR/antagonist or CAR/inverse agonist complexes. A salt bridge between the CoR and a conserved lysine in helix 4 of the NR is central to this interaction, but also helix 12 was stabilized by direct contacts with residues of the CoR. Fixation of helix 12 in the antagonistic/inverse agonistic conformation prevents an energetically unfavorable free floatation of the C terminus. The comparable molecular mechanisms that explain the similar functional profile of antagonist and inverse agonists are likely to be extended from VDR and CAR to other members of the NR superfamily and may lead to the design of even more effective ligands.     

Critical role of helix 12 of the vitamin D(3) receptor for the partial agonism of carboxylic ester antagonists.

The carboxy-terminal alpha-helix of a nuclear receptor ligand-binding domain (LBD), helix 12, contains a critical, ligand-modulated interface for the interaction with coactivator proteins. In this study, using the example of the vitamin D receptor (VDR) and the partial antagonist ZK159222, the role of helix 12 (residues 417-427) for both antagonistic and agonistic receptor actions was investigated. Amino acid residue G423 was demonstrated to be critical for partial agonism of ZK159222, but not for the activity of the natural VDR agonist, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). The amount of partial agonism of ZK159222 increased when helix 12 was truncated by the last four amino acid residues (Delta424-27) and augmented even more, when in addition helix 12 of VDR's dimerization partner, retinoid X receptor (RXR), was truncated. In contrast, the low agonism of a structural derivative of ZK159222, ZK168281, was not affected comparably, whereas other close structural relatives of ZK159222 even demonstrated the same agonistic activity as that of 1alpha,25(OH)(2)D(3). The amount of agonism of ZK159222 and ZK168281 at different variations of helix 12 correlated well with VDR's ability to complex with coactivator proteins and inversely correlated with the strength of the compound's antagonistic action on 1alpha,25(OH)(2)D(3) signalling. Molecular dynamics simulations of the LBD complexed with the two antagonists could explain their different action by demonstrating a more drastic displacement of helix 12 through ZK168281 than through ZK159222. Moreover, the modelling could indicate a kink of helix 12 at amino acid residue G423, which provides the last four amino acid residues of helix 12 with a modulatory role for the partial agonism of some VDR antagonists, such as ZK159222. In conclusion, partial agonism of a VDR antagonist is lower the more it disturbs helix 12 in taking the optimal position for coactivator interaction.     

Molecular basis of the selective activity of vitamin D analogues

More than 2,000 synthetic analogues of the biological active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), are presently known. Basically, all of them interfere with the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signaling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR), and a 1alpha,25(OH)(2)D(3) response element (VDRE). Central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilized by a 1alpha,25(OH)(2)D(3) analogue either in its agonistic, antagonistic, or non-agonistic conformation. The positioning of helix 12 of the LBD is of most critical importance for these conformations. In each of the three conformations, the VDR performs different protein-protein interactions, which then result in a characteristic functional profile. Most 1alpha,25(OH)(2)D(3) analogues have been identified as agonists, a few are antagonists (e.g., ZK159222 and TEI-9647), and only Gemini and some of its derivatives act under restricted conditions as non-agonists. The functional profile of some 1alpha,25(OH)(2)D(3) analogues, such as EB1089 and Gemini, can be modulated by protein and DNA interaction partners of the VDR. This provides them with some selectivity for DNA-dependent and -independent signaling pathways and VDRE structures.     

Central role of VDR conformations for understanding selective actions of vitamin D(3) analogues

The vitamin D(3) receptor (VDR) acts primarily as a heterodimer with the retinoid X receptor (RXR) on different types of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) response elements (VDREs). Therefore, DNA-bound VDR-RXR heterodimers can be considered as the molecular switches of 1alpha,25(OH)(2)D(3) signalling. Functional conformations of the VDR within these molecular switches appear to be of central importance for describing the biologic actions of 1alpha,25(OH)(2)D(3) and its analogues. Moreover, VDR conformations provide a molecular basis for understanding the potential selective profile of VDR agonists, which is critical for a therapeutic application. This review discusses VDR conformations and their selective stabilization by 1alpha,25(OH)(2)D(3) and its analogues, such as EB1089 and Gemini, as a monomer in solution or as a heterodimer with RXR bound to different VDREs and complexed with coactivator or corepressor proteins.     

Structural determinants for vitamin D receptor response to endocrine and xenobiotic signals

The vitamin D receptor (VDR), initially identified as a nuclear receptor for 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], regulates calcium metabolism, cellular proliferation and differentiation, immune responses, and other physiological processes. Recently, secondary bile acids such as lithocholic acid (LCA) were identified as endogenous VDR agonists. To identify structural determinants required for VDR activation by 1alpha,25(OH)2D3 and LCA, we generated VDR mutants predicted to modulate ligand response based on sequence homology to pregnane X receptor, another bile acid-responsive nuclear receptor. In both vitamin D response element activation and mammalian two-hybrid assays, we found that VDR-S278V is activated by 1alpha,25(OH)2D3 but not by LCA, whereas VDR-S237M can respond to LCA but not to 1alpha,25(OH)2D3. Competitive ligand binding analysis reveals that LCA, but not 1alpha,25(OH)2D3, effectively binds to VDR-S237M and both 1alpha,25(OH)2D3 and LCA bind to VDR-S278V. We propose a docking model for LCA binding to VDR that is supported by mutagenesis data. Comparative analysis of the VDR-LCA and VDR-1alpha,25(OH)2D3 structure-activity relationships should be useful in the development of bile acid-derived synthetic VDR ligands that selectively target VDR function in cancer and immune disorders without inducing adverse hypercalcemic effects.     

A novel mutation in helix 12 of the vitamin D receptor impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder most often caused by mutations in the vitamin D receptor (VDR). The patient in this study exhibited the typical clinical features of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated serum concentrations of alkaline phosphatase and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D(3)]. The patient did not have alopecia. Assays of the VDR showed a normal high affinity low capacity binding site for [(3)H]1,25-(OH)(2)D(3) in extracts from the patient's fibroblasts. However, the cells were resistant to 1,25-dihydroxyvitamin D action as demonstrated by the failure of the patient's cultured fibroblasts to induce the 24-hydroxylase gene when treated with either high doses of 1,25-(OH)(2)D(3) or vitamin D analogs. A novel point mutation was identified in helix H12 in the ligand-binding domain of the VDR that changed a highly conserved glutamic acid at amino acid 420 to lysine (E420K). The patient was homozygous for the mutation. The E420K mutant receptor recreated by site-directed mutagenesis exhibited many normal properties including ligand binding, heterodimerization with the retinoid X receptor, and binding to vitamin D response elements. However, the mutant VDR was unable to elicit 1,25-(OH)(2)D(3)-dependent transactivation. Subsequent studies demonstrated that the mutant VDR had a marked impairment in binding steroid receptor coactivator 1 (SRC-1) and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex. Taken together, our data indicate that the mutation in helix H12 alters the coactivator binding site preventing coactivator binding and transactivation. In conclusion, we have identified the first case of a naturally occurring mutation in the VDR (E420K) that disrupts coactivator binding to the VDR and causes HVDRR.     

Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide

We have determined the crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor in ternary complexes with a synthetic LXXLL-containing peptide and the following four ligands: 1alpha,25-dihydroxyvitamin D(3); 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD); 1alpha-hydroxy-2-methylene-19-nor-(20S)-bishomopregnacalciferol (2MbisP), and 2alpha-methyl-19-nor-1alpha,25-dihydroxyvitamin D(3) (2AM20R). The conformation of the LBD is identical in each complex. Binding of the 2-carbon-modified analogues does not change the positions of the amino acids in the ligand binding site and has no effect on the interactions in the coactivator binding pocket. The CD ring of the superpotent analogue, 2MD, is tilted within the binding site relative to the other ligands in this study and to (20S)-1alpha,25-dihydroxyvitamin D(3) [Tocchini-Valentini et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 5491-5496]. The aliphatic side chain of 2MD follows a different path within the binding site; nevertheless, the 25-hydroxyl group at the end of the chain occupies the same position as that of the natural ligand, and the hydrogen bonds with histidines 301 and 393 are maintained. 2MbisP binds to the receptor despite the absence of the 25-hydroxyl group. A water molecule is observed between His 301 and His 393 in this structure, and it preserves the orientation of the histidines in the binding site. Although the alpha-chair conformer is highly favored in solution for the A ring of 2AM20R, the crystal structures demonstrate that this ring assumes the beta-chair conformation in all cases, and the 1alpha-hydroxyl group is equatorial. The peptide folds as a helix and is anchored through hydrogen bonds to a surface groove formed by helices 3, 4, and 12. Electrostatic and hydrophobic interactions between the peptide and the LBD stabilize the active receptor conformation. This stablization appears necessary for crystal growth.     

Crystal structure of human estrogen-related receptor alpha in complex with a synthetic inverse agonist reveals its novel molecular mechanism

Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).     

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)?D?], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D? [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D? [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.     

Gene regulatory potential of nonsteroidal vitamin D receptor ligands

The seco-steroid 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is a promising drug candidate due to its pleiotropic function including the regulation of calcium homeostasis, bone mineralization and cellular proliferation, differentiation, and apoptosis. We report here a novel class of nonsteroidal compounds, represented by the bis-aromatic molecules CD4409, CD4420, and CD4528, as ligands of the 1alpha,25(OH)2D3 receptor (VDR). Taking the known diphenylmethane derivative LG190178 as a reference, this study provides molecular evaluation of the interaction of nonsteroidal ligands with the VDR. All four nonsteroidal compounds were shown to induce VDR-retinoid X receptor heterodimer complex formation on a 1alpha,25(OH)2D3 response element, stabilize the agonistic conformation of the VDR ligand-binding domain, enable the interaction of VDR with coactivator proteins and contact with their three hydroxyl groups the same residues within the ligand-binding pocket of the VDR as 1alpha,25(OH)2D3. Molecular dynamics simulations demonstrated that all four nonsteroidal ligands take a shape within the ligand-binding pocket of the VDR that is very similar to that of the natural ligand. CD4528 is mimicking the natural hormone best and was found to be in vitro at least five times more potent than LG190178. In living cells, CD4528 was only two times less potent than 1alpha,25(OH)2D3 and induced mRNA expression of the VDR target gene CYP24 in a comparable fashion. At a noncalcemic dose of 150 microg/kg, CD4528 showed in vivo a clear induction of CYP24 expression and therefore may be used as a lead compound for the development of therapeutics against psoriasis, osteoporosis, and cancer.     

Synthesis of 1alpha,25-dihydroxyvitamin D3-26,23-lactams (DLAMs), a novel series of 1 alpha,25-dihydroxyvitamin D3 antagonist

Novel vitamin D(3) analogs having a lactam structure in their side chains, 1 alpha,25-dihydroxyvitamin D(3)-26,23-lactams (DLAMs), were designed based on the principle of regulation of the folding of helix-12 in the vitamin D nuclear receptor (VDR). The new analogs were synthesized via 1,3-dipolar cycloaddition reaction and subsequent reduction of the isoxazolidine as key steps. Among the analogs, (23S,25S)-DLAM-01 (4a) and (23S,25S)-DLAM-1P (5a) bind strongly to VDR. Moreover, these analogs were found to inhibit the differentiation of HL-60 cells induced by 1 alpha,25-dihydroxyvitamin D(3).