Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element

The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27 gene promoter. The conformational changes in the protein-DNA during the formation of the UspDBD-EcRDBD-hsp27 complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5'- and/or 3'-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5'-end of the regulatory element and moved close to the 3'-end. An analysis was also done on the effect that DHR38DBD, the Drosophila ortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27 bending was induced by DHR38DBD. Fluorescence data revealed that hsp27 had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27 in the presence of EcRDBD is presented.     

Polarity of the ecdysone receptor complex interaction with the palindromic response element from the hsp27 gene promoter.

The functional 20-hydroxyecdysone (20E) receptor is a heterodimer of two members of the nuclear hormone receptors superfamily; the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. As most of the natural 20E-response elements are highly degenerated palindromes, we were interested in determining whether or not such asymmetric elements could dictate the defined orientation of the Usp/EcR complex. We have investigated interaction of EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) with the palindromic response element from the hsp27 gene promoter (hsp27pal). The hsp27pal half-sites contribute differently to the binding of the heterodimer components; the 5' half-site exhibits higher affinity for both DBDs than the 3' half-site. This observation, along with data demonstrating that UspDBD exhibits approximate fourfold higher affinity to the 5' half-site than EcRDBD, suggest that UspDBD locates the EcRDBD/UspDBD heterocomplex in the defined orientation (5'-UspDBD-EcRDBD-3') on the hsp27pal sequence. The binding polarity onto hsp27pal is accompanied by different contribution of the UspDBD and EcRDBD C-terminal sequences to the DNA-binding and heterocomplex formation. This is supported by finding that deletion of the C-terminal of EcRDBD region corresponding to the putative A-helix severely decreased binding of the EcRDBD to the hsp27pal. In contrast, UspDBD in which corresponding residues were deleted exhibited the same hsp27pal binding pattern as the wild type UspDBD. Additional truncation comprising the putative T-box, resulted in a reduced binding of the mutated UspDBD. This truncation however, still allowed effective EcRDBD/UspDBD heterodimer formation. Finally we demonstrated that perfect palindromes, composed of two hsp27pal 5' half-sites (or of the related sequence) contain all of the structural information necessary for the anisotropic UspDBD/EcRDBD heterocomplex formation. However, the perfect palindromes bind isolated homomeric DBDs as well as their heterocomplex with higher affinity than imperfect hsp27pal. This is the first report indicating that natural 20E response elements, which with one exception are degenerated palindromes, may act as functionally asymmetric elements in a manner similar to the action of direct repeats in vertebrates.     

Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response element.

The steroid hormone, 20-hydroxyecdysone (20E), directs Drosophila metamorphosis via a heterodimeric receptor formed by two members of the nuclear hormone receptors superfamily, the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. Our previous study [Niedziela-Majka, A., Kochman, M., Ozyhar, A. (2000) Eur. J. Biochem. 267, 507-519] on EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) suggested that UspDBD may act as a specific anchor that preferentially binds the 5' half-site of the pseudo-palindromic response element from the hsp27 gene promoter and thus locates the heterocomplex in the defined orientation. Here, we analyzed in detail the determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition alpha helix and the roles of the base pairs of the UspDBD target sequence have been probed by site-directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocomplex. It is suggested how possible nucleotide deviations within the 5' half-site of the element may be used for the fine-tuning of the 20E-response element specificity and consequently the physiological response.     

The application of an immobilized molecular beacon for the analysis of the DNA binding domains from the ecdysteroid receptor proteins Usp and EcR's interaction with the hsp27 response element

The nonstandard molecular beacon described in this article consists of 2 fragments, each built of a short single-stranded oligonucleotide sequence and a double-stranded sequence. One of these hybridization probes, labeled with a fluorescence donor (fluorescein), is solid phase immobilized. The second nonimmobilized probe is labeled with a fluorescence quencher (dabcyl). Annealing of both probes via single-stranded sequences was possible only in the presence of a specific protein molecule that recognized the response element sequence initially separated between the immobilized and nonimmobilized fragments. The system was applied successfully to detect the sequence-specific interaction of a natural hsp27 response element from the promoter of the hsp27 gene with the DNA binding domains of 2 nuclear receptor proteins: ultraspiracle Usp (UspDBD) and the ecdysone receptor EcR (EcRDBD). Measured in the absence of EcRDBD, the dissociation constant, K(d) of the UspDBD-hsp27 complex, was determined to be 3.26 nM, whereas for UspDBD devoid of the A-box (UspDBDDeltaA-hsp27 ), the dissociation constant was 4.81 nM. The respective K(d) values in the presence of EcRDBD were 2.43 nM and 10.80 nM. The results obtained with the immobilized molecular beacon technology were in agreement with those obtained by conventional fluorescence titrations and by fluorescence resonance energy transfer measurements with nonimmobilized beacons.     

The Molecular Basis of Conformational Instability of the Ecdysone Receptor DNA Binding Domain Studied by In Silico and In Vitro Experiments

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule.     

Plasticity of the ecdysone receptor DNA binding domain

Ecdysteroids coordinate molting and metamorphosis in insects via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and the ultraspiracle (Usp) protein. Here we show how the DNA-recognition alpha-helix and the T box region of the EcR DNA-binding domain (EcRDBD) contribute to the specific interaction with the natural response element and to the stabilization of the EcRDBD molecule. The data indicate a remarkable mutational tolerance with respect to the DNA-binding function of the EcRDBD. This is particularly manifested in the heterocomplexes formed between the EcRDBD mutants and the wild-type Usp DNA-binding domain (UspDBD). Circular dichroism (CD) spectra and protein unfolding experiments indicate that, in contrast to the UspDBD, the EcRDBD is characterized by a lower alpha-helix content and a lower stability. As such, the EcRDBD appears to be an intrinsically unstructured protein-like molecule with a high degree of intramolecular plasticity. Because recently published crystal structures indicate that the ligand binding domain of the EcR is also characterized by the extreme adaptability, we suggest that plasticity of the EcR domains may be a key factor that allows a single EcR molecule to mediate diverse biological effects.     

Dual FRET assay for detecting receptor protein interaction with DNA

We present here a new assay that is based on the idea of the molecular beacon. This assay makes it possible to investigate two proteins interacting with DNA at two binding sites that are close to each other. The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors. As a model we used the components of the heterodimeric ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR) from Drosophila melanogaster and a response element from the promoter of the hsp27 gene. The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs). We have shown that protein–protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27_pal response element. The analysis of the microscopic dissociation constants obtained with the DMB led to the conclusion that there was increased affinity of UspDBD to the 5′ half-site in the presence of EcRDBD when the 3′ half-site was occupied, and increased affinity of EcRDBD to the 3′ half-site when the 5′ half-site was occupied.     

Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95Å structure of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD, which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part of the C-terminal extension (CTE) of the EcRDBD folds into an α-helix whose location in the minor groove does not match any of the locations previously observed for nuclear receptors. Mutational analyses suggest that the α-helix is a component of EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to be a general feature of all known EcRs.     

The composite nature of the interaction between nuclear receptors EcR and DHR38

Ecdysteroids coordinate essential biological processes in Drosophila through a complex of two nuclear receptors, the ecdysteroid receptor (EcR) and the ultraspiracle protein (Usp). Biochemical experiments have shown that, in contrast to Usp, the EcR molecule is characterized by high intramolecular plasticity. To investigate whether this plasticity is sufficient to form EcR complexes with nuclear receptors other than Usp, we studied the interaction of EcR with the DHR38 nuclear receptor. Previous in vitro experiments suggested that DHR38 can form complexes with Usp and thus disrupt Usp-EcR interaction with the specific hsp27pal response element. This article provides the experimental evidence that EcR is able to form complexes with DHR38 as well. The recombinant DNA-binding domains (DBDs) of EcR and DHR38 interact specifically on hsp27pal. However, the interaction between the receptors is not restricted to their isolated DBDs. We pre\xadsent data that indicate that the full-length EcR and DHR38 can also form specific complexes within the nuclei of living cells. This interaction is mediated by the hinge region of EcR, which was recently classified as an intrinsically disordered region. Our results indicate that DHR38 might modulate the activity of the Usp-EcR heterodimer by forming complexes with both of its components.     

Purification of Drosophila melanogaster ultraspiracle protein and analysis of its A/B region-dependent dimerization behavior in vitro

Two members of the nuclear receptor superfamily, EcR (ecdysteroid receptor protein) and Usp (Ultraspiracle), heterodimerize to form a functional receptor for the steroid hormone 20-hydroxyecdysone and thus enable it to coordinate morphogenetic events during insect metamorphosis. N-terminally His-tagged Usp was overexpressed in E. coli cells as a non-truncated protein and purified to homogeneity in two chromatographic steps. It was demonstrated that the recombinant receptor specifically binds the ecdysone response element of the hsp27 gene promoter (hsp27EcRE). Moreover, a highly synergistically formed heterodimeric complex with the DNA-binding domain of EcR was observed on hsp27EcRE, but not on the native Usp response element from the chorion s15 gene promoter. Recombinant Usp forms homodimers and homotetramers in the absence of DNA, as judged from gel filtration and chemical crosslinking experiments. Truncation of its N-terminal A/B region changes molecular characteristics of Usp, considerably weakening its oligomerization potential under the same experimental conditions. This contrasts with the results obtained previously for the similarly truncated RXR--a vertebrate homolog of Usp.     

DNA bending induced by high mobility group proteins studied by fluorescence resonance energy transfer.

The HMG domains of the chromosomal high mobility group proteins homologous to the vertebrate HMG1 and HMG2 proteins preferentially recognize distorted DNA structures. DNA binding also induces a substantial bend. Using fluorescence resonance energy transfer (FRET), we have determined the changes in the end-to-end distance consequent on the binding of selected insect counterparts of HMG1 to two DNA fragments, one of 18 bp containing a single dA(2) bulge and a second of 27 bp with two dA(2) bulges. The observed changes are consistent with overall bend angles for the complex of the single HMG domain with one bulge and of two domains with two bulges of approximately 90-100 degrees and approximately 180-200 degrees, respectively. The former value contrasts with an inferred value of 150 degrees reported by Heyduk et al. (1) for the bend induced by a single domain. We also observe that the induced bend angle is unaffected by the presence of the C-terminal acidic region. The DNA bend of approximately 95 degrees observed in the HMG domain complexes is similar in magnitude to that induced by the TATA-binding protein (80 degrees), each monomeric unit of the integration host factor (80 degrees), and the LEF-1 HMG domain (107 degrees). We suggest this value may represent a steric limitation on the extent of DNA bending induced by a single DNA-binding motif.     

Impact of heterodimerization on intracellular localization of the ecdysteroid receptor (EcR)

Initially, nuclear import of the ecdysteroid receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (Usp(R130C)), which has lost its DNA binding capability. The C-terminal end of Usp (Usp(Delta205-508)) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way.     

Tertiary conformation of the template-primer and gapped DNA substrates in complexes with rat polymerase beta. Fluorescence energy transfer studies using the multiple donor-acceptor approach

The tertiary structure of template-primer and gapped DNA substrates in the complex with rat polymerase beta (pol beta) has been examined using the fluorescence energy transfer method based on the multiple donor-acceptor approach. In these studies, we used DNA substrates labeled at the 5' end of the template strand and the 5' end of the primer with the fluorescent donor and/or acceptor. Measurements of the enzyme complex with the template-primer DNA substrate having a ten nucleotide long ssDNA extension indicate that the distance between the 5' end of the template strand and the 5' end of the primer decreases by approximately 9.8 A as compared to the free nucleic acid. Analogous experiments with the template-primer substrate, having the ssDNA extension with five nucleotide residues, show approximately 6.6 A distance decrease. Such large distance decreases indicate that the DNA is significantly bent in the binding site. Analysis of the data indicates that the bending occurs between the third and the fourth nucleotide of the ssDNA extension. The entire template strand is at the bend angle Theta(TP) = 85 +/- 7 degrees with respect to the dsDNA part of the DNA molecule. In the polymerase complex with the gapped DNA, the distance between the 5' ends of the DNA and the bend angle are 66 +/- 2.2 A and 65 +/- 6 degrees, respectively. These values are very similar to the same distance and bend angle of the gap complex in the crystal structure of the co-complex. The presence of the 5'-terminal PO(4)(-) group downstream from the primer does not affect the tertiary conformation of the gapped DNA, indicating that the effect of the phosphate group is localized at the ssDNA gap.     

Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle

The Ecdysone receptor (EcR) is distributed between cytoplasm and nucleus in CHO cells. Nuclear localization is increased by the ligand Muristerone A. The most important heterodimerization partner Ultraspiracle (Usp) is localized predominantly in the nucleus. We used the diethylentriamine nitric oxide adduct DETA/NO, which releases NO and destroys the zinc-finger structure of nuclear receptors, to investigate whether nuclear EcR and Usp interact with DNA. If expressed separately, Usp and EcR in the absence of hormone do not interact with DNA. The hormone-induced increase in nuclear EcR is due to enhanced DNA binding. In the presence of Usp, EcR is shifted nearly quantitatively into the nucleus. Only a fraction (approximately 30%) of the heterodimer is sensitive to DETA/NO. Interaction of the heterodimer with DNA is mediated mainly by the C-domain of EcR. Deletion of the DNA-binding domain of Usp only slightly reduces nuclear localization of EcR/Usp, although the nuclear localization signal of Usp is not present anymore. The results indicate that EcR and Usp can enter the nucleus independently, but cotransport of both receptors mediated by dimerization via the ligand binding domains is possible even in the absence of hormone.     

Nucleocytoplasmic shuttling of the ecdysteroid receptor (EcR) and of ultraspiracle (Usp) from Drosophila melanogaster in mammalian cells: energy requirement and interaction with exportin

The small G protein Ran, which is important for nucleocytoplasmic shuttling of proteins is present, but does not interact with EcR, Usp, and EcR/Usp. As shown by oligomycin treatment, EcR, Usp, and EcR/Usp import is energy dependent. Export of EcR and EcR/Usp is mediated by exportin-1 (CRM-1) as shown by the inhibiting effect of leptomycin B (LMB). Usp remains in the nucleus for more than 24 h. Nuclear retainment of EcR and Usp is energy dependent as shown by treatment with oligomycin. No export signal could be identified for Usp. The data confirm that EcR and Usp can enter the nucleus independently and that intracellular localization is regulated individually for each receptor. It is also demonstrated that the export signal of EcR is inaccessible after heterodimerization with Usp.     

HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study

HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA. In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements. A static bend was achieved by the introduction of two phased A4T4 tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 microM(-1)). The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend. DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor. From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated. The end-to-end distance relative to DNA contour length (R/R(C)) yields a bend angle for the A-tract duplex of 45 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU. The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex. These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism.     

DNA binding properties of the ecdysteroid receptor in the salivary gland of the female ixodid tick, Amblyomma hebraeum

Salivary gland degeneration in the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae) is controlled by an ecdysteroid hormone. In an earlier study (Mao, H., McBlain, W.A., Kaufman, W.R., 1995. Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum. Gen. Comp. Endocrinol. 99, 340–348), we demonstrated that a protein component of a salivary gland extract binds to ponasterone A (Pon A) with high affinity (Kd1 nM), suggesting a tick ecdysteroid receptor (EcR). In this study, the Pon A binding protein bound to calf thymus DNA; this binding could be dissociated by Drosophila hsp27 EcRE. The binding protein shifted the [³²P]hsp27 EcRE band on a gel mobility shift assay; formation of the complex with hsp27 EcRE required KCl (optimal concentration was approximately 75 mM). A number of physiologically effective ecdysteroids enhanced the binding with the following order of potency: Pon A>Mur A>Mak A>20E>ecdysone, whereas vertebrate steroids (estradiol, cholesterol, corticosterone, progesterone, testosterone) had no such effect. Using monoclonal antibodies against Drosophila EcR and USP, we found that AG10.2 recognized three bands (90.5, 87.3 and 84 kDa for EcR) and AB11 recognized at least two major bands (50.3 and 47.1 kDa for USP) in the salivary gland extract by western blot analysis. In addition, AB11 supershifted the tick EcR-hsp27 EcRE band on a gel mobility shift assay, indicating that the tick EcR heterodimerized with a USP-like protein for DNA binding. Furthermore, selective mutations to the 15-basepair palindrome of hsp27 EcRE at positions −5, +2, or adding a base to the spacer, resulted in considerably reduced affinity to the tick EcR/USP. We thus propose a sequence similarity of EcREs between A. hebraeum and its insect counterpart.