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There is an increasing need for rapid, reliable, non-invasive, and inexpensive mass testing methods as the global COVID-19 pandemic continues. Detection dogs could be a possible solution to identify individuals infected with SARS-CoV-2. Previous studies have shown that dogs can detect SARS-CoV-2 on sweat samples. This study aims to establish the dogs' sensitivity (true positive rate) which measures the proportion of people with COVID-19 that are correctly identified, and specificity (true negative rate) which measures the proportion of people without COVID-19 that are correctly identified. Seven search and rescue dogs were tested using a total of 218 axillary sweat samples (62 positive and 156 negative) in olfaction cones following a randomised and double-blind protocol. Sensitivity ranged from 87% to 94%, and specificity ranged from 78% to 92%, with four dogs over 90%. These results were used to calculate the positive predictive value and negative predictive value for each dog for different infection probabilities (how likely it is for an individual to be SARS-CoV-2 positive), ranging from 10-50%. These results were compared with a reference diagnostic tool which has 95% specificity and sensitivity. Negative predictive values for six dogs ranged from ≥98% at 10% infection probability to ≥88% at 50% infection probability compared with the reference tool which ranged from 99% to 95%. Positive predictive values ranged from ≥40% at 10% infection probability to ≥80% at 50% infection probability compared with the reference tool which ranged from 68% to 95%. This study confirms previous results, suggesting that dogs could play an important role in mass-testing situations. Future challenges include optimal training methods and standardisation for large numbers of detection dogs and infrastructure supporting their deployment., Competing Interests: The authors have declared that no competing interests exist.

The insect steroid hormone 20-hydroxyecdysone (20E) binds to its cognate nuclear receptor composed of the ecdysone receptor (EcR) and Ultraspiracle (USP) and triggers the main developmental transitions, in particular molting and metamorphosis. We present the crystal structure of the ligand-binding domains of EcR/USP in complex with 20E at 2.4A resolution and compare it with published structures of EcR/USP bound to ponasterone A (ponA). ponA is essentially identical to 20E but lacks the 25-OH group of 20E. The structure of 20E-bound EcR indicates that an additional hydrogen bond is formed compared with the ponA-bound receptor, yet, paradoxically, ponA has a significantly higher affinity for EcR than 20E. Theoretical studies based on docking and free energy methods lead to a rationale for understanding the difference in binding affinities between 20E and ponA. Results of the calculations indicate that the favorable contribution from the extra H-bond made by 25-OH of 20E is counterbalanced by its larger desolvation cost compared with that of ponA. The contribution of 25-OH to the binding affinity is further compared with those of 20- and 22-OH groups. Ligands that lack the 20- or 22-OH group are indeed known to bind less favorably to EcR than 20E, an effect opposite to that observed for ponA. The results indicate that their respective contributions to receptor-ligand complex stability reside mostly in their different contributions to solvation/desolvation. Together, the data demonstrate the critical role of ligand desolvation in determining binding affinity, with general implications for the binding of hormones to their cognate nuclear receptors.

Purpose: To investigate functional consequence on photoreceptor-cell specific nuclear receptor (NR2E3) transcriptional activity of enhanced S-cone syndrome (ESCS) mutations localized in ligand binding domain (LBD)., Methods: Point mutations were introduced into the LBD of full length and Gal4 chimeric NR2E3 receptors and transcriptional activity was investigated by using transient co-transfection assay on corresponding luciferase reporters. Expression and DNA binding properties of transfected mutant and wild-type receptors were tested by Western blotting and gel shift assay., Results: Our analysis show that two ESCS mutations, missense mutations R385P and M407K, abolished NR2E3 repressive activity in the context of full-length and Gal4 chimeric receptors, while W234S and R311Q mutants retained their repressive activity in both assays. All mutant receptors maintained their stability and DNA binding ability., Conclusions: These results showed that NR2E3 mutations localized in LBD induce ESCS disease without affecting inhibitory activity as recorded in vitro. This demonstrates the absence of correlation between transcriptional inhibition and ESCS phenotype. This analysis suggests that NR2E3 might have transcriptional activation properties not yet identified.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-alpha (ERalpha) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERalpha in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERalpha remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERalpha ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERalpha monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERalpha ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.

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.

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., (Copyright 2004 The National Academy of Sciencs of the USA)

SmFtz-F1 (Schistosoma mansoni Fushi Tarazu-Factor 1) belongs to the Ftz-F1 subfamily of nuclear receptors, but displays marked structural differences compared with its mammalian homologues SF-1 (steroidogenic factor-1) or liver receptor homologue-1. These include a long F domain (104 amino acids), an unusually large hinge region (133 amino acids) and a poorly conserved E-domain. Here, using Gal4 constructs and a mammalian two-hybrid assay, we have characterized the roles of these specific regions both in the transcriptional activity of the receptor and in its interactions with cofactors. Our results have shown that, although the AF-2 (activation function-2) region is the major activation function of the receptor, both the F and D domains are essential for AF-2-dependent activity. Modelling of SmFtz-F1 LBD (ligand-binding domain) and structure-guided mutagenesis allowed us to show the important role of helix H1 in maintaining the structural conformation of the LBD, and suggested that its autonomous transactivation activity, also observed with SF-1, is fortuitous. This strategy also allowed us to study an eventual ligand-dependence for this orphan receptor, the predicted three-dimensional models suggesting that the SmFtz-F1 LBD contains a large and well-defined ligand-binding pocket sealed by two arginine residues orientated towards the interior of the cavity. Mutation of these two residues provoked a loss of transcriptional activity of the receptor, and strongly reduced its interaction with SRC1 (steroid receptor cofactor-1), suggesting a ligand-dependent activity for SmFtz-F1. Taken together, our results argue for original and specific functional activities for this platyhelminth nuclear receptor.

Dinoflagellates are marine unicellular eukaryotes that exhibit unique features including a very low level of basic proteins bound to the chromatin and the complete absence of histones and nucleosomal structure. A cDNA encoding a protein with a strong homology to the TATA box-binding proteins (TBP) has been isolated from an expressed sequence tag library of the dinoflagellate Crypthecodinium cohnii. The typical TBP repeat signature and the amino acid motives involved in TFIIA and TFIIB interactions were conserved in this new TBP-like protein. However, the four phenylalanines known to interact with the TATA box were substituted with hydrophilic residues (His(77), Arg(94), Tyr(171), Thr(188)) as has been described for TBP-like factors (TLF)/TBP-related proteins (TRP). A phylogenetic analysis showed that cTBP is intermediate between TBP and TLF/TRP protein families, and the structural similarity of cTBP with TLF was confirmed by low affinity binding to a consensus' TATA box in an equivalent manner to that usually observed for TLFs. Six 5'-upstream gene regions of dinoflagellate genes have been analyzed and neither a TATA box nor a consensus-promoting element could be found within these different sequences. Our results showed that cTBP could bind stronger to a TTTT box sequence than to the canonical TATA box, especially at high salt concentration. Same binding results were obtained with a mutated cTBP (mcTBP), in which the four phenylalanines were restored. To our knowledge, this is the first description of a TBP-like protein in a unicellular organism, which also appears as the major form of TBP present in C. cohnii.

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor 3 (ERR3) complexed with a steroid receptor coactivator-1 (SRC-1) peptide reveals a transcriptionally active conformation in absence of any ligand. The structure explains why estradiol does not bind ERRs with significant affinity. Docking of the previously reported ERR antagonists, diethylstilbestrol and 4-hydroxytamoxifen, requires structural rearrangements enlarging the ligand binding pocket that can only be accommodated with an antagonist LBD conformation. Mutant receptors in which the ligand binding cavity is filled up by bulkier side chains still interact with SRC-1 in vitro and are transcriptionally active in vivo, but are no longer efficiently inactivated by diethylstilbestrol or 4-hydroxytamoxifen. These results provide structural and functional evidence for ligand-independent transcriptional activation by ERR3.

The ultraspiracle protein (USP) is the insect ortholog of the mammalian retinoid X receptor (RXR). Fundamental questions concern the functional role of USP as the heterodimerization partner of insect nuclear receptors such as the ecdysone receptor. The crystallographic structures of the ligand binding domain of USPs of Heliothis virescens and Drosophila melanogaster solved recently show that helix 12 is locked in an antagonist conformation raising the question whether USPs could adopt an agonist conformation as observed in RXRalpha. In order to investigate this hypothesis, a homology model for USP is proposed that allows a structural analysis of the agonist conformation of helix 12 based on the sequence comparison with RXR. For USP, one of the main issues concerns its function and in particular whether its activity is ligand independent or not. The x-ray structures strongly suggest that USP can bind ligands. Putative ligands have therefore been docked in the USP homology model. Juvenile hormones and juvenile hormone analogs were chosen as target ligands for the docking study. The interaction between the ligand and the receptor are examined in terms of the pocket shape as well as in terms of the chemical nature of the residues lining the ligand binding cavity.

The retinoic acid-related orphan receptor beta (RORbeta) exhibits a highly restricted neuronal-specific expression pattern in brain, retina and pineal gland. So far, neither a natural RORbeta target gene nor a functional ligand have been identified, and the physiological role of the receptor is not well understood. We present the crystal structure of the ligand-binding domain (LBD) of RORbeta containing a bound stearate ligand and complexed with a coactivator peptide. In the crystal, the monomeric LBD adopts the canonical agonist-bound form. The fatty acid ligand-coactivator peptide combined action stabilizes the transcriptionally active conformation. The large ligand-binding pocket is strictly hydrophobic on the AF-2 side and more polar on the beta-sheet side where the carboxylate group of the ligand binds. Site-directed mutagenesis experiments validate the significance of the present structure. Homology modeling of the other isotypes will help to design isotype-selective agonists and antagonists that can be used to characterize the physiological functions of RORs. In addition, our crystallization strategy can be extended to other orphan nuclear receptors, providing a powerful tool to delineate their functions.

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.

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.

Androgens, like progestins, are 3-ketosteroids with structural differences restricted to the 17beta substituent in the steroid D-ring. To better understand the specific recognition of ligands by the human androgen receptor (hAR), a homology model of the ligand-binding domain (LBD) was constructed based on the progesterone receptor LBD crystal structure. Several mutants of residues potentially involved in the specific recognition of ligands in the hAR were constructed and tested for their ability to bind agonists. Their transactivation capacity in response to agonist (R1881) and antagonists (cyproterone acetate, hydroxyflutamide, and ICI 176344) was also measured. Substitution of His(874) by alanine, only marginally impairs the ligand-binding and transactivation capacity of the hAR receptor. In contrast, mutations of Thr(877) and, to a greater extent, Asn(705) perturb ligand recognition, alter transactivation efficiency, and broaden receptor specificity. Interestingly, the N705A mutant acquires progesterone receptor (PR) properties for agonist ligands but, unlike wild type AR and PR, loses the capacity to repress transactivation with nonsteroidal antagonists. Models of the hAR.LBD complexes with several ligands are presented, which suggests new directions for drug design.

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.

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.

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.

The crystal structure of a heterodimer between the ligand-binding domains (LBDs) of the human RARalpha bound to a selective antagonist and the constitutively active mouse RXRalphaF318A mutant shows that, pushed by a bulky extension of the ligand, RARalpha helix H12 adopts an antagonist position. The unexpected presence of a fatty acid in the ligand-binding pocket of RXRalpha(F318A is likely to account for its apparent "constitutivity." Specific conformational changes suggest the structural basis of pure and partial antagonism. The RAR-RXR heterodimer interface is similar to that observed in most nuclear receptor (NR) homodimers. A correlative analysis of 3D structures and sequences provides a novel view on dimerization among members of the nuclear receptor superfamily.

The action of 1 alpha, 25-dihydroxyvitamin D3 is mediated by its nuclear receptor (VDR), a ligand-dependent transcription regulator. We report the 1.8 A resolution crystal structure of the complex between a VDR ligand-binding domain (LBD) construct lacking the highly variable VDR-specific insertion domain and vitamin D. The construct exhibits the same binding affinity for vitamin D and transactivation ability as the wild-type protein, showing that the N-terminal part of the LBD is essential for its structural and functional integrity while the large insertion peptide is dispensable. The structure reveals the active conformation of the bound ligand and allows understanding of the different binding properties of some synthetic analogs.

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.

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.

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.

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.

Key residues of the human mineralocorticoid receptor (hMR) involved in the recognition of agonist and antagonist ligands were identified by alanine-scanning mutagenesis based on a homology model of the hMR ligand-binding domain. They were tested for their transactivation capacity and ability to bind agonists (aldosterone, cortisol) and antagonists (progesterone, RU26752). The three-dimensional model reveals two polar sites located at the extremities of the elongated hydrophobic ligand-binding pocket. Mutations of Gln776 and Arg817 in site I reduce the affinity of hMR for both agonists and antagonists and affect the capacity of hMR to activate transcription, suggesting that the C3-ketone group, common to all ligands, is anchored by these two residues conserved within the nuclear steroid receptor family. In contrast, mutations of Asn770 and Thr945 in the opposite site only affect the binding of agonists bearing the C21-hydroxyl group. The binding of hMR antagonists that exhibit a smaller size and faster off-rate kinetics compared with agonists is not affected. In the light of the hMR homology model, a new mechanism of antagonism is proposed in which the AF2-AD core region is destabilized by the loss of contacts between the antagonist and the helix H12 region.

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.

Mutations of a single residue in the retinoid X receptor alpha (RXRalpha) ligand-binding pocket (LBP) generate constitutive, ligand-binding-competent mutants with structural and functional characteristics similar to those of agonist-bound wild-type RXR. Modelling of the mouse RXRalphaF318A LBP suggests that, like agonist binding, the mutation disrupts a cluster of van der Waals interactions that maintains helix H11 in the apo-receptor location, thereby shifting the thermodynamic equilibrium to the holo form. Heterodimerization with some apo-receptors (retinoic acid, thyroid hormone and vitamin D3 receptors) results in 'silencing' of RXRalphaF318A constitutive activity, which, on the other hand, efficiently contributes to synergistic transactivation within NGFI-B-RXR heterodimers. RAR mutants disabled for corepressor binding and/or lacking a functional AF-2 activation domain, do not relieve RXR 'silencing'. Not only RAR agonists, but also the RAR antagonist BMS614 induce conformational changes allowing RXR to exert constitutive (RXRalphaF318A) or agonist-induced (wild-type RXR) activity in heterodimers. Interestingly, the RXRalphaF318A constitutive activity generated within heterodimers in the presence of BMS614 requires the integrity of both RXR and RAR AF-2 domains. These observations suggest that, within RXR-RAR heterodimers, RAR can adopt a structure distinct from that of the active holo-RAR, thus allowing RXR to become transcriptionally responsive to agonists.

The crystal structure of human cyclin H refined at 2.6 A resolution is compared with that of cyclin A. The core of the molecule consists of two repeats containing five helices each and forming the canonical cyclin fold also observed in TFIIB. One hundred and thirty-two out of the 217 C alpha atoms from the cyclin fold can be superposed with a root-mean-square difference of 1.8 A. The structural homology is even higher for the residues at the interface with the kinase, which is of functional significance, as shown by our observation that cyclin H binds to cyclin-dependent kinase 2 (cdk2) and that cyclin A is able to activate cdk7 in the presence of MAT1. Based on this superposition, a new signature sequence for cyclins was found. The specificity of the cyclin H molecule is provided mainly by two long helices which extend the cyclin fold at its N- and C-termini and pack together against the first repeat on the side opposite to the kinase. Deletion mutants show that the terminal helices are required for a functionally active cyclin H.

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.