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.     

Corepressor excess shifts the two-side chain vitamin D analog Gemini from an agonist to an inverse agonist of the vitamin D receptor

The vitamin D receptor (VDR) is an endocrine nuclear receptor that binds with high affinity its natural ligand 1alpha,25-dihydroxyvitamin D3. Gemini is a 1alpha,25-dihydroxyvitamin D3 analog with two identical side chains that, despite its significantly increased volume, binds to the VDR and can function as a potent agonist. This study demonstrates that, at excess corepressor (CoR) levels, Gemini shifts from an agonist to an inverse agonist that actively recruits CoR proteins to the VDR and mediates superrepression. Under these conditions Gemini stabilizes the VDR into a silent conformation, in which helix 12 of the ligand-binding domain is repositioned and thus unable to contribute to coactivator interaction. Amino acid F422 has been described as the lock of helix 12 and seems to be the most critical VDR residue in the inverse agonistic action of Gemini. Molecular dynamics simulations of the Gemini-VDR complex support these observation by indicating that the second side chain of Gemini induces tension to the receptor structure that can be released by a shift of helix 12. Taken together, Gemini is the first described (conditional) inverse agonist to an endocrine nuclear receptor and may function as a sensor for the cell-specific coactivator/CoR ratio.     

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.     

The critical role of carboxy-terminal amino acids in ligand-dependent and -independent transactivation of the constitutive androstane receptor

The mouse constitutive androstane receptor (CAR) is a unique member of the nuclear receptor superfamily, for which an inverse agonist, the testosterone metabolite 5alpha-androstan-3alpha-ol (androstanol), and an agonist, the xenobiotic 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene, are known. In this study the role of the transactivation domain 2 (AF-2) of CAR was investigated, which is formed by the seven most carboxy-terminal amino acids of the receptor. The AF-2 domain was shown to be critical for the constitutive activity by mediating a ligand-independent interaction of CAR with coactivator (CoA) proteins. In addition this domain increased and decreased contact with CoAs in the presence of agonist and inverse agonist, respectively. In analogy to classical endocrine nuclear receptors, in CAR the charge clamp between K187 (in helix 3) and E355 (within the AF-2 domain) was expected to be critical for its interaction with CoAs. However, the hydrophobic amino acids L352, L353, and I356 on the surface of the AF-2 domain were found to be more important for this protein-protein interaction. Moreover, these amino acids and C357 were shown to be involved in the response of CAR to androstanol. Interestingly, the cysteine at position 357 appears to block classical endocrine responsiveness of CAR to agonists, since mutagenesis of this amino acid both reduced CoA interaction in the absence of ligand and drastically increased inducibility by 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene. We showed that this blockade is not due to an intramolecular disulfide bridge, but is probably caused by an interaction between C357 and Y336.     

TR surfaces and conformations required to bind nuclear receptor corepressor

Residues of the TR that are critical for binding the nuclear receptor corepressor (N-CoR) were identified by testing more than 100 separate mutations of the full-length human TRbeta that scan the surface of its ligand binding domain. The primary inferred interaction surface overlaps the surface described for binding of p160 coactivators, but differs by extending to a novel site underneath which helix 12 rests in the liganded TR, rather than including residues of helix 12. Nonconservative mutations of this surface diminished binding similarly to three isolated N-CoR receptor interaction domains (RIDs), but conservative mutations affected binding variably, consistent with a role for this surface in RID selectivity. The commonality of this surface in binding N-CoR was confirmed for the RXRs and ERs. Deletion of helix 12 increased N-CoR binding by the TR modestly, and by the RXR and ER to a much greater extent, indicating a competition between this helix and the corepressor that regulates the extent of corepressor binding by nuclear receptors. When helix 12 was deleted, N-CoR binding by the ER was stimulated by tamoxifen, and binding by the TR was stimulated by Triac, indicating that helix 12 is not the only feature that regulates corepressor binding. Two additional mutationsensitive surfaces were found alongside helix 1, near the previously described CoR box, and above helix 11, nearby but separate from residues that help link receptor in dimers. Based on effects of selected mutations on T(3) and coactivator binding, and on results of combined mutations of the three sites on corepressor binding, we propose that the second and third surfaces stabilize TR unliganded conformation(s) required for efficient N-CoR binding. In transfection assays mutations of all three surfaces impaired the corepressor-mediated functions of unliganded TR repression or activation. These detailed mapping results suggest approaches for selective modulation of corepressor interaction that include the shape of the molecular binding surface, the competitive occupancy by helix 12, pharmacological stimulation, and specific conformational stabilization.     

Agonist-dependent and agonist-independent transactivations of the human constitutive androstane receptor are modulated by specific amino acid pairs

The constitutive androstane receptor (CAR) is an interesting member of the nuclear receptor superfamily because of its exceptionally high constitutive activity due to ligand-independent interaction of the ligand-binding domain with co-activator proteins. This study compares the agonist-dependent and agonist-independent activities of human CAR with those of mouse CAR and the vitamin D receptor and demonstrates that the constitutive activity of CAR is mediated by at least three contacts between the amino acids of helix 12, partner amino acids in helices 4 and 11, and a charge clamp between helices 12 and 3. The stabilization of helix 12 by a contact between its C terminus and the lysine of helix 4 has the same impact in human and mouse CARs. In addition, the charge clamp between the glutamate in helix 12 and the lysine in helix 3 is also important for the constitutive activity of both receptor orthologs but less critical for the agonist-dependent stabilization of their respective helices 12. Interestingly, Cys-357 in mouse CAR has significantly more impact on the stabilization of helix 12 than does the orthologous position Cys-347 in human CAR. This deficit appears to be compensated by a more dominant role of Ile-330 in human CAR over Leu-340 in mouse CAR because it is more efficient than Cys-347 in controlling the flexibility of helix 12 in the presence of an agonist. The constitutive activity of other members of the nuclear receptor superfamily could be explained by a homologous hydrophobic interaction between large, non-polar amino acids of helices 11 and 12.     

Structural determinants of the agonist-independent association of human peroxisome proliferator-activated receptors with coactivators

Lipid homeostasis is controlled by various nuclear receptors (NRs), including the peroxisome proliferator-activated receptors (PPARalpha, delta, and gamma), which sense lipid levels and regulate their metabolism. Here we demonstrate that human PPARs have a high basal activity and show ligand-independent coactivator (CoA) association comparable with the NR constitutive androstane receptor. Using PPARgamma as an example, we found that four different amino acid groups contribute to the ligand-independent stabilization of helix 12 of the PPAR ligand-binding domain. These are: (i) Lys329 and Glu499, mediating a charge clamp-type stabilization of helix 12 via a CoA bridge; (ii) Glu352, Arg425, and Tyr505, directly stabilizing the helix via salt bridges and hydrogen bonds; (iii) Lys347 and Asp503, interacting with each other as well as contacting the CoA; and (iv) His351, Tyr(355), His477, and Tyr501, forming a hydrogen bond network. These amino acids are highly conserved within the PPAR subfamily, suggesting that the same mechanism may apply for all three PPARs. Phylogenetic trees of helix 12 amino acid and nucleotide sequences of all crystallized NRs and all human NRs, respectively, indicated a close relationship of PPARs with constitutive androstane receptor and other constitutive active members of the NR superfamily. Taking together, the ligand-independent tight control of the position of the PPAR helix 12 provides an effective alternative for establishing an interaction with CoA proteins. This leads to high basal activity of PPARs and provides an additional view on PPAR signaling.     

Meclizine is an agonist ligand for mouse constitutive androstane receptor (CAR) and an inverse agonist for human CAR

The constitutive androstane receptor (CAR, NR1I3) is a key regulator of xenobiotic and endobiotic metabolism. The ligand-binding domains of murine (m) and human (h) CAR are divergent relative to other nuclear hormone receptors, resulting in species-specific differences in xenobiotic responses. Here we identify the widely used antiemetic meclizine (Antivert; Bonine) as both an agonist ligand for mCAR and an inverse agonist for hCAR. Meclizine increases mCAR transactivation in a dose-dependent manner. Like the mCAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, meclizine stimulates binding of steroid receptor coactivator 1 to the murine receptor in vitro. Meclizine administration to mice increases expression of CAR target genes in a CAR-dependent manner. In contrast, meclizine suppresses hCAR transactivation and inhibits the phenobarbital-induced expression of the CAR target genes, cytochrome p450 monooxygenase (CYP)2B10, CYP3A11, and CYP1A2, in primary hepatocytes derived from mice expressing hCAR, but not mCAR. The inhibitory effect of meclizine also suppresses acetaminophen-induced liver toxicity in humanized CAR mice. These results demonstrate that a single compound can induce opposite xenobiotic responses via orthologous receptors in rodents and humans.