The TBP-like factor: an alternative transcription factor in metazoa?

Protein sequence analysis has revealed a family of TATA-binding-protein (TBP)-like factors (TLFs) in metazoan organisms. Modelling of the three-dimensional structure of these TLFs suggests that they form an asymmetric saddle-like structure and that, unlike TBP, TLFs might bind to DNA sequences other than classical TATA boxes. Thus, the existence of TLFs presents a challenge to the doctrine that TBP is a universal regulator of transcription in metazoans.

Human TAF(II)55 interacts with the vitamin D(3) and thyroid hormone receptors and with derivatives of the retinoid X receptor that have altered transactivation properties.

We have identified novel interactions between the human (h)TATA-binding protein-associated factor TAF(II)55 and the ligand-binding domains (LBDs) of the nuclear receptors for vitamin D(3) (VDR) and thyroid hormone (TRalpha). Following expression in Cos cells, hTAF(II)55 interacts with the VDR and TRalpha LBDs in a ligand-independent manner whereas no interactions with the retinoid X receptors (RXRs) or with other receptors were observed. Deletion mapping indicates that hTAF(II)55 interacts with a 40-amino-acid region spanning alpha-helices H3 to H5 of the VDR and TRalpha LBDs but not with the equivalent highly related region of RXRgamma. TAF(II)55 also interacts with chimeric receptors in which the H3-to-H5 region of RXRgamma has been replaced with that of the VDR or TRalpha. Furthermore, replacement of two single amino acids of the RXRgamma LBD with their VDR counterparts allows the RXRgamma LBD to interact with hTAF(II)55 while the corresponding double substitution allows a much stronger interaction. In transfection experiments, the single mutated RXRgamma LBDs activate transcription to fivefold higher levels than wild-type RXRgamma while the double mutation activates transcription to a level comparable to that observed with the VDR. There is therefore a correlation between the ability of the modified RXRs to interact with hTAF(II)55 and transactivation. These results strongly suggest that the TAF(II)55 interactions with the modified RXR LBDs modulate transcriptional activation.

Characterization of the DNA-binding and dimerization properties of the nuclear orphan receptor germ cell nuclear factor.

The orphan receptor germ cell nuclear factor (GCNF) is a member of the superfamily of nuclear receptors. During development, GCNF exhibits a restricted brain-specific expression pattern, whereas GCNF expression in the adult is germ cell specific. Therefore, the receptor may participate in the regulation of neurogenesis and reproductive functions. No natural GCNF target gene has yet been identified, but recent data demonstrate specific and high-affinity binding of GCNF either to the direct repeat DNA element AGGTCAAGGTCA (DR0) or to extended half-sites, such as TCAAGGTCA. In this study, we show that murine GCNF (mGCNF) can bind as a homodimer to extended half-sites, thus describing a novel property within the nuclear receptor superfamily. Homodimeric binding to extended half-sites requires the presence of a dimerization function within the mGCNF DNA-binding domain (DBD) and a novel dimerization surface encompassing the putative helix 3 and the helix 12 region of the mGCNF ligand-binding domain (LBD). In addition, the mGCNF LBD has the potential to adopt different conformations with distinct dimerization properties. The helix 12 region of the mGCNF LBD not only regulates the switch between these dimerization conformations but also dictates the DNA-binding behavior and transcriptional properties of the different dimerization conformations. In summary, our findings describe unique DNA-binding and dimerization properties of a nuclear receptor and suggest a novel mechanism that allows mGCNF to modulate target gene activity.

Investigation of the binding interactions of progesterone using muteins of the human progesterone receptor ligand binding domain designed on the basis of a three-dimensional protein model.

The aim of this study was to investigate the binding interactions of the human progesterone receptor (hPR) with its natural ligand. Therefore, a homology-derived model of the hPR ligand binding domain has been constructed and used to predict residues potentially involved in interactions with progesterone. These residues and the free cysteines have been mutated (in total 13 residues with 15 mutations). All exchanges have been designed to preserve the three-dimensional structure of the protein. With respect to the binding characteristics towards progesterone, the muteins fall into three groups displaying no, reduced, or wildtype-like binding activity.

Structural basis for engineering of retinoic acid receptor isotype-selective agonists and antagonists.

BACKGROUND: Many synthetic retinoids have been generated that exhibit a distinct pattern of agonist/antagonist activities with the three retinoic acid receptors (RARalpha, RARbeta and RARgamma). Because these retinoids are selective tools with which to dissect the pleiotropic functions of the natural pan-agonist, retinoic acid, and might constitute new therapeutic drugs, we have determined the structural basis of their receptor specificity and compared their activities in animal and yeast cells. RESULTS: There are only three divergent amino acid residues in the ligand binding pockets (LBPs) of RARalpha, RARbeta and RARgamma. We demonstrate here that the ability of monospecific (class I) retinoid agonists and antagonists to bind to and induce or inhibit transactivation by a given isotype is directly linked to the nature of these residues. The agonist/antagonist potential of class II retinoids, which bind to all three RARs but depending on the RAR isotype have the potential to act as agonists or antagonists, was also largely determined by the three divergent LBP residues. These mutational studies were complemented by modelling, on the basis of the three-dimensional structures of the RAR ligand-binding domains, and a comparison of the retinoid agonist/antagonist activities in animal and yeast cells. CONCLUSIONS: Our results reveal the rational basis of RAR isotype selectivity, explain the existence of class I and II retinoids, and provide a structural concept of ligand-mediated antagonism. Interestingly, the agonist/antagonist characteristics of retinoids are not conserved in yeast cells, suggesting that yeast co-regulators interact with RARs in a different way than the animal cell homologues do.

Crucial role of the H11-H12 loop in stabilizing the active conformation of the human mineralocorticoid receptor.

The crystal structures of ligand-free and agonist-associated ligand-binding domain (LBD) of nuclear receptors (NRs) reveal that the amphipathic helix H12 is folded back toward the LBD core in the agonist-associated conformation, allowing the binding of coactivators. We used alanine scanning mutagenesis to explore the role of the residues of the loop connecting H11 and H12 in the activation of the human mineralocorticoid receptor (hMR), a member of the NRs family. H950A retained the ligand binding and transcriptional activities of the wild-type receptor and interacted with coactivators. In contrast F956A had no receptor functions. Aldosterone bound to the mutant hMRs (L952A, K953A, V954A, E955A, P957A) with nearly the same affinity as to the wild-type receptor and caused a receptor conformational change in these mutant hMRs as it does for the wild-type receptor. But the aldosterone-induced transcriptional activity of the mutant hMRs was lower (L952A, E955A, P957A) than that of the wild-type receptor or completely abolished (K953A, V954A) and their interaction with coactivators was impaired (E955A) or suppressed (L952A, K953A, V954A, P957A). In the light of a hMR-LBD model based on the structure of the progesterone-associated receptor-LBD, we propose that the integrity of the H11-H12 loop is crucial for folding the receptor into a ligand-binding competent state and for establishing the network of contacts that stabilize the active receptor conformation.

A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita.

The DAX-1 gene encodes an unusual member of the nuclear hormone receptor superfamily. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. We have shown that DAX-1 binds to hairpin secondary structures and blocks steroidogenesis in adrenal cells via transcriptional repression of the steroidogenic acute regulatory protein (StAR) promoter. Here we have investigated the molecular mechanism of DAX-1-mediated repression. We show that the DAX-1 C terminus contains a potent transcriptional silencing activity, which can be transferred to a heterologous DNA-binding domain. Deletion analysis and modeling of DAX-1 structure identify two cooperating domains required for the silencing function, one located within helix H3 and the other within H12. The silencing function is cell- and promoter-specific. Strikingly, two point mutations (R267P and deltaV269) found in adrenal hypoplasia patients impair silencing. These findings suggest that transcriptional silencing by DAX-1 plays a critical role in the pathogenesis of adrenal hypoplasia congenita.

Residues in the ligand binding domain that confer progestin or glucocorticoid specificity and modulate the receptor transactivation capacity.

To localize regions conferring ligand binding specificity of the human glucocorticoid (hGR) and progesterone (hPR) receptors, we constructed chimeras comprising the DNA-binding domain of the yeast transcription factor GAL4, linked to the ligand binding domain of hGR or hPR. Replacement of a sequence of hGR encompassing helices H6 and H7 with the homologous sequence from hPR creates a chimeric protein GP3, which binds the progestin RU 27987 with high affinity, and results in a concomitant loss of glucocorticoid binding [dexamethasone (DEX), RU 43044]. Moreover, GP3 is not able to mediate RU 27987-induced transactivation. A detailed mutational analysis of this sequence and the study of the recently solved hPR crystal structure revealed five residues that confer progestin responsiveness to GR or modulate ligand binding and transcriptional activation. Notably, the simultaneous presence of residues Ser637 and Phe639 on GP3, lining the ligand binding pocket, is specifically involved in RU 27987 binding. The absence of residues Asp641, Gln642, and Leu647 on GP3 is accountable for the lack of glucocorticoids binding on GP3. Unlike residue 642, residues 641 and 647 are not in direct contact with the ligand and most likely act through steric-mediated interactions. The presence of Gln642 and Leu647 are determinant for transcriptional activation in response to DEX and RU 27987, respectively. DEX-dependent transactivation is further enhanced by the presence of Leu647.

A new model for 20-hydroxyecdysone and dibenzoylhydrazine binding: a homology modeling and docking approach.

The ecdysone receptor (ECR), a nuclear transcription factor controlling insect development, is a novel target for insecticides such as dibenzoylhydrazines with low environmental and toxicological impacts. To understand the high selectivity of such synthetic molecules toward ECR, two homology models of the Chironomus tentans ECR ligand-binding domain (LDB) have been constructed by taking as templates the known LBD crystal structures of the retinoic acid and vitamin D receptors. Docking of 20-hydroxyecdysone (20E) and dibenzoylhydrazines to the receptor suggests a novel superposition of the natural and synthetic molecules; the N-tert-butyl substituent of the dibenzoylhydrazines extends significantly beyond the 20E volume. Our ECR-LBD protein models rationalize how 20E and dibenzoylhydrazines interact with the ligand-binding pocket. The homology model complexes provide new insights that can be exploited in the rational design of new environmentally safe insecticides.

Sequences in the ligand-binding domains of the human androgen and progesterone receptors which determine their distinct ligand identities.

The natural ligands of the progesterone (PR) and androgen (AR) receptors, progesterone and testosterone, differ only by their 17 beta-substitution. To identify within the AR and PR ligand-binding domains (LBDs) the sequences responsible for the differential recognition of these ligands, chimeric LBDs assembled from five homologous AR/PR 'cassettes' linked to the GAL4-DNA binding domain were constructed, and their ligand binding and transactivation characteristics were determined. Replacing the central cassette 3 of PR by that of AR generated a progesterone- and testosterone-responsive PR LBD with the AR residues 788-RHLS-791 being specifically involved in testosterone recognition, while the introduction of the C-terminal PR cassette 5 into AR conferred progestin responsiveness onto the AR LBD. These results suggest that residues within AR 788-RHLS-791 interact with the testosterone 17 beta-OH, while PR cassette 5 apparently contains the amino acid(s) specifically involved in the recognition of the progesterone 17 beta-acetyl group. However, ligand binding and transactivation by these chimeras were significantly decreased compared with those of the parental LBDs, indicating that residues located outside of these cassettes contribute to the proper positioning of the steroids in the AR and PR ligand-binding pockets (LBPs). Indeed, certain AR/PR chimeras acquired efficient ligand binding, but were unable to transactivate, indicating that the ligand was improperly bound in the chimeric. LBP and could not induce the conformational changes leading to a transcriptionally competent activation function (AF-2) within the LBD. The properties of the various LBD chimeras are discussed in view of the recently solved three-dimensional structures of the retinoid X receptor alpha apo- and retinoic acid receptor gamma holo-LBDs.

Three-dimensional models of estrogen receptor ligand binding domain complexes, based on related crystal structures and mutational and structure-activity relationship data.

On the basis of the recently determined crystal structures of the ligand binding domains (LBDs) of the retinoic acid nuclear receptors (NRs), we present a three-dimensional (3D) molecular model of the human estrogen receptor alpha (hERalpha) LBD. A literature search for mutants affecting the binding properties has been performed; 45 out of 48 published mutants can be explained satisfactorily on the basis of the model. Estradiol has been docked into the binding pocket to probe its interactions with the protein. Energy minimizations and molecular dynamics calculations were performed for various ligand orientations. To evaluate their quality, the different models were scored using known structure-activity relationship (SAR) data for selected close estradiol homologues. The two best models explain largely the binding affinities of more distantly related ligands.

Unfaithfulness and promiscuity of a mutant androgen receptor in a hormone-refractory prostate cancer.

Missense mutations in the androgen receptor (AR) contribute to the failure of hormonal therapy for prostate cancer (PCa), but the underlying molecular bases remain uncharacterized. Here, we describe a new AR variant found in a hormone-refractory metastatic PCa, in which threonine 575 in the DNA binding domain, and threonine 877 in the ligand-binding domain, were both replaced by an alanine. Using gene reporter assays, we demonstrate that the T575A mutation weakened transcriptional activity from promoters containing AR-specific responsive elements, while activity from promoters with AR-non-specific elements was enhanced. Data from gel shift experiments revealed a preferential binding of the T575A mutant to AR-non-specific motifs. We demonstrate that the two mutations T575A and T877A cooperate to confer new functional properties on the AR, and that the mutant AR functions simultaneously as a promiscuous AR due to the T877A mutation, and an unfaithful AR due to the T575A mutation.

Ligand selectivity by nuclear hormone receptors.

Abstract Numerous crystal structures of nuclear receptor ligand binding domains (LBDs) are known. The retinoic acid (RAR) and estrogen (ER) receptors are the two members for which a large set of agonists and antagonist complexes are available. Their analysis reveals key features of the RAR and ER ligand binding pocket (LBP) responsible for ligand selectivity. The RAR LBD exhibits a rigid architecture to which the ligand has to adapt, whereas the ER LBD can accomodate numerous ligands of variable shapes.

Net (ERP/SAP2) one of the Ras-inducible TCFs, has a novel inhibitory domain with resemblance to the helix-loop-helix motif.

The three ternary complex factors (TCFs), Net (ERP/ SAP-2), ELK-1 and SAP-1, are highly related ets oncogene family members that participate in the response of the cell to Ras and growth signals. Understanding the different roles of these factors will provide insights into how the signals result in coordinate regulation of the cell. We show that Net inhibits transcription under basal conditions, in which SAP-1a is inactive and ELK-1 stimulates. Repression is mediated by the NID, the Net Inhibitory Domain of about 50 amino acids, which autoregulates the Net protein and also inhibits when it is isolated in a heterologous fusion protein. Net is particularly sensitive to Ras activation. Ras activates Net through the C-domain, which is conserved between the three TCFs, and the NID is an efficient inhibitor of Ras activation. The NID, as well as more C-terminal sequences, inhibit DNA binding. Net is more refractory to DNA binding than the other TCFs, possibly due to the presence of multiple inhibitory elements. The NID may adopt a helix-loop-helix (HLH) structure, as evidenced by homology to other HLH motifs, structure predictions, model building and mutagenesis of critical residues. The sequence resemblance with myogenic factors suggested that Net may form complexes with the same partners. Indeed, we found that Net can interact in vivo with the basic HLH factor, E47. We propose that Net is regulated at the level of its latent DNA-binding activity by protein interactions and/or phosphorylation. Net may form complexes with HLH proteins as well as SRF on specific promotor sequences. The identification of the novel inhibitory domain provides a new inroad into exploring the different roles of the ternary complex factors in growth control and transformation.

Estrogen receptor transcription and transactivation: Structure-function relationship in DNA- and ligand-binding domains of estrogen receptors.

Estrogen receptors are members of the nuclear receptor steroid family that exhibit specific structural features, ligand-binding domain sequence identity and dimeric interactions, that single them out. The crystal structures of their DNA-binding domains give some insight into how nuclear receptors discriminate between DNA response elements. The various ligand-binding domain crystal structures of the two known estrogen receptor isotypes (alpha and beta) allow one to interpret ligand specificity and reveal the interactions responsible for stabilizing the activation helix H12 in the agonist and antagonist positions.

Different ligands-different receptor conformations: modeling of the hER alpha LBD in complex with agonists and antagonists.

The aim of this study is to compare crystal structures of nuclear receptor ligand binding domains in complex with different agonists and partial agonists to achieve a better understanding of the three-dimensional structures and their ligand-induced conformational changes. This led to the identification of structurally conserved "rigid" regions and more flexible parts of the proteins. The analysis was found to be of great value in fitting selected non-steroidal compounds into the human estrogen receptor alpha (hER alpha) ligand binding pocket. The experimentally determined binding affinities for a number of 2-aryl indoles and 2-aryl indenones are in good agreement with the subsequently modeled binding interactions. To date, no crystal structure is published for a complex with a pure antagonist. We therefore used the available structural information on complexes with partial agonists and the crystal structure of a mutant protein in complex with estradiol displaying a similar conformation to predict binding interactions for antagonists. The results are discussed in detail.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands.

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1alpha,25(OH)(2)D(3) and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17beta-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

Crystal structure of a mutant hERalpha ligand-binding domain reveals key structural features for the mechanism of partial agonism.

The crystal structure of a triple cysteine to serine mutant ERalpha ligand-binding domain (LBD), complexed with estradiol, shows that despite the presence of a tightly bound agonist ligand, the protein exhibits an antagonist-like conformation, similar to that observed in raloxifen and 4-hydroxytamoxifen-bound structures. This mutated receptor binds estradiol with wild type affinity and displays transcriptional activity upon estradiol stimulation, but with limited potency (about 50%). This partial activity is efficiently repressed in antagonist competition assays. The comparison with available LBD structures reveals key features governing the positioning of helix H12 and highlights the importance of cysteine residues in promoting an active conformation. Furthermore the present study reveals a hydrogen bond network connecting ligand binding to protein trans conformation. These observations support a dynamic view of H12 positioning, where the control of the equilibrium between two stable locations determines the partial agonist character of a given ligand.