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A hybrid system made of gold nanorods (AuNRs) and double-stranded DNA (dsDNA) is used to build a versatile multi-logic gate platform, capable of performing six different logic operations. The sequence-specific binding of transcription factors to the DNA drives the optical response of the design.

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. Recent live cell imaging studies have revealed that interactions of GR with chromatin are highly dynamic, with average receptor residence times of only seconds. These findings were surprising because early kinetic studies found that GR-DNA interactions in vitro were much slower, having calculated residence times of minutes to hours. However, these latter analyses were conducted at a time when it was possible to work with only either partially purified holoreceptor or its purified but isolated DNA binding domain. Noting these limitations, we reexamined GR-DNA dissociation kinetics using a highly purified holoreceptor shown to be amenable to rigorous study. We first observe that GR-DNA interactions in vitro are not slow as previously thought but converge with in vivo behavior, having residence times of only seconds to tens of seconds. This rapid exchange is seen at six individual response elements and the multisite MMTV promoter used in live cell imaging. Second, GR dissociation rates are identical for all response elements. Thus, previously observed differences in receptor affinity toward these sequences are not due to differences in off rate but in on rate. Finally, dissociation kinetics are biphasic in character. A minimal kinetic model consistent with the data is that in which DNA-bound GR interconverts between states on a second time scale, with dissociation occurring via a multistep process. We speculate that receptor interconversion in this time frame can be recognized by the coregulatory proteins that interact with GR, leading to unique transcriptional responses.

To thoroughly understand the role that estrogen receptors partake in regulation of gene expression, characterization of estrogen receptors (ERs) and estrogen-response elements (EREs) interactions is essential. In the work, we present a highly sensitive and reusable silicon nanowire (SiNW) biosensor to study the interactions between human ER proteins (ER, α and β subtypes) and EREs (dsDNA). The proteins were covalently immobilized on the SiNW surface. Various EREs including wild-type, mutant and scrambled DNA sequences were then applied to the protein-functionalized SiNW surface. Due to negatively charged dsDNA, binding of the EREs to the ERs on the n-type SiNW biosensor leads to the accumulation of negative charges on the surface, thereby inducing increase in resistance. The results show that the specificity of the ERE-ERα binding is higher than that of the ERE-ERβ binding, what is more, the mutant ERE reduces the binding affinity for both ERα and ERβ. By applying various concentrations of wild-type ERE to the bound ERα, a very low concentration of 10 fM wild-type ERE was found to be able to bind to the ERα. The reversible association and dissociation between ERα and wt-ERE was achieved, pointing to a reusable biosensor for protein-DNA binding. Through the study, we have established the SiNW biosensor as a promising method in providing comprehensive study for hormone receptor-response element interactions., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Molecular orbital calculations of the complex between DNA-ERE (estrogen response element) and ER (estrogen receptor)-DBD (DNA-binding domain) were performed using the fragment molecular orbital (FMO) method, which enables large-scale MO (molecular orbital) calculations by reducing the computational cost and by significantly increasing efficiency for parallel computation. Such a large system, which contains 3354 atoms, is impractical via conventional MO methods due to the immense computational cost. Details of the interaction between DNA-ERE and ER-DBD were revealed in this study as follows by using the FMO calculations to analyze the interfragment interaction energies (IFIEs) and the electrostatic potentials (ESPs). An area with a high positive ESP is identified on the DNA-binding side of ER-DBD and is the main driving force behind access to the DNA. The position of the ER-DBD monomer can be fixed on a phosphate group of DNA-ERE by the strong electrostatic interactions, whereas the rotation cannot be fixed. In contrast, both the position and rotation of the ER-DBD dimer can be fixed and can therefore form the stable (ER-DBD)2...DNA-ERE complex. Dimerization of the ER-DBD monomers, each of which have a charge of +5 , is mainly due to large attractive interaction energies of the second Zn fragments. The base pairs in the consensus sequence of DNA-ERE interact only with the recognition helix located in the major groove due to the large shielding effect of the phosphate groups of DNA. The recognition helix has weaker interactions with the base pairs than the electrostatic interactions with the phosphate groups. Thus, the DNA-binding machinery of the ER-DBD dimer, which can secure the recognition helix in the major groove of DNA, is crucial for interactions between the recognition helix and base pairs.

HMGB-1/-2 are coregulatory proteins that facilitate the DNA binding and transcriptional activity of steroid receptor members of the nuclear receptor family of transcription factors. We investigated the influence and mechanism of action of HMGB-1/-2 (formerly known as HMG-1/-2) on estrogen receptor alpha (ERalpha) and ERbeta. Both ER subtypes were responsive to HMGB-1/-2 with respect to enhancement of receptor DNA binding affinity and transcriptional activity in cells. Responsiveness to HMGB-1/-2 was dependent on the C-terminal extension (CTE) region of the ER DNA binding domain (DBD) and correlated with a direct protein interaction between HMGB-1/-2 and the CTE. Thus the previously reported higher DNA binding affinity and transcription activity of ERalpha as compared with ERbeta is not due to a lack of ERbeta interaction with HMGB-1/-2. Using chimeric receptor DBDs, the higher intrinsic DNA binding affinity of ERalpha than ERbeta was shown to be due to a unique property of the ERalpha CTE, independent of HMGB-1/-2. The CTE of both ER subtypes was also shown to be required for interaction with ERE half-sites. These studies reveal the importance of the CTE and HMGB-1/-2 for ERalpha and ERbeta interaction with their cognate target DNAs.

A fractal analysis of a confirmative nature only is presented for the binding of estrogen receptor (ER) in solution to its corresponding DNA (estrogen response element, ERE) immobilized on a sensor chip surface [J. Biol. Chem. 272 (1997) 11384], and for the cooperative binding of human 1,25-dihydroxyvitamin D(3) receptor (VDR) to DNA with the 9-cis-retinoic acid receptor (RXR) [Biochemistry 35 (1996) 3309]. Ligands were also used to modulate the first reaction. Data taken from the literature may be modeled by using a single- or a dual-fractal analysis. Relationships are presented for the binding rate coefficient as a function of either the analyte concentration in solution or the fractal dimension that exists on the biosensor surface. The binding rate expressions developed exhibit a wide range of dependence on the degree of heterogeneity that exists on the surface, ranging from sensitive (order of dependence equal to 1.202) to very sensitive (order of dependence equal to 12.239). In general, the binding rate coefficient increases as the degree of heterogeneity or the fractal dimension of the surface increases. The predictive relationships presented provide further physical insights into the reactions occurring on the biosensor surface. Even though these reactions are occurring on the biosensor surface, the relationships presented should assist in understanding and in possibly manipulating the reactions occurring on cellular surfaces.

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.

Estrogen receptors (ERs) efficiently potentiate the transcriptional activity of prolactin-activated Stat5b through a mechanism that involves the ER DNA-binding domain (DBD) and the hinge domain. We have identified residues within the DBD of ER that are critical for the functional interaction of ER with Stat5b. We show that disruption of the second zinc finger structure abrogated cross-talk between ER and Stat5b, while the structure of the first zinc finger was not important. Furthermore, we confirm that intact DNA binding activity was not required for potentiation of Stat5b activity and that the dimerization of ER did not seem to be involved. Ligand-bound ERs also modulated activating protein 1-dependent transcription, and our data demonstrate that both zinc finger structures of the ER DBD are important for an intact response. We show that introduction of various point mutations within the DBD altered the response of the receptor to 17beta-estradiol and to the estrogen antagonists 4-hydroxytamoxifen and ICI 182,870 on the collagenase promoter. These findings provide new insights into the mechanisms by which ERs act in cross-talk with non-related transcription factors.

The nuclear/hormone receptors are an extensive family of ligand-activated transcription factors that recognise DNA targets through a highly conserved, structurally autonomous DNA-binding domain. The compact structure of the DNA-binding domain is supported by two zinc ions, each of which is co-ordinated by the tetrahedral arrangement of thiol groups from four cysteine residues. Metal binding is expected to be linked with deprotonation of the co-ordinating thiol groups and folding of the polypeptide. Using a variety of biophysical approaches, we characterise these linked equilibria for the isolated DNA-binding domains (DBD) of the receptors for estrogen and glucocorticoid. Mass spectrometry and equilibrium denaturation indicate that, near neutral pH, approximately four of the eight co-ordinating thiol groups release protons with zinc uptake, in agreement with the expected pK(a) change for the -SH group in the presence of the metal. Mass spectrometry reveals that the protein charge distribution changes with the uptake of zinc and that metal binding is co-operative. The co-operativity is consistent with observations from equilibrium denaturation, which indicate that the folding event is a two-state process. A crucial residue that stabilises the equilibrium structure of the DBD fold itself is a cysteine residue situated in the hydrophobic core of all known nuclear hormone receptors (but not involved in metal binding): it appears to be conserved absolutely for its unique combination of size and hydrophobicity. Stabilisation of the DBDs could be achieved by truncating the flexible, basic termini, suggesting that like-charge clusters may have deleterious effects on protein folds. While the metal-free apo protein and the chemically denatured state have little defined secondary structure, these states were expanded only partially in comparison with the native structure, according to data from small-angle X-ray scattering. The comparatively compact shapes of the denatured and apo forms may explain, in part, the marginal stability of the native fold.

Estrogen acting through the estrogen receptor (ER) is able to regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors. Ligand-activated ER binds DNA and transactivates the promoters of estrogen target genes. In addition, ligand-activated ER can interact with other factors to alter the physiology and growth of cells. Using a yeast two-hybrid screen, we have identified an interaction between ER alpha and the proapoptotic forkhead transcription factor FKHR. The ER alpha-FKHR interaction depends on beta-estradiol and is reduced significantly in the absence of hormone or the presence of Tamoxifen. A glutathione S-transferase pull-down assay was used to confirm the interaction and localized two interaction sites, one in the forkhead domain and a second in the carboxyl terminus. The FKHR interaction was specific to ER alpha and was not detected with other ligand-activated steroid receptors. The related family members, FKHRL1 and AFX, also bound to ER alpha in the presence of beta-estradiol. FKHR augmented ER alpha transactivation through an estrogen response element. Conversely, ER alpha repressed FKHR-mediated transactivation through an insulin response sequence, and cell cycle arrest induced by FKHRL1 in MCF7 cells was abrogated by estradiol. These results suggest a novel mechanism of estrogen action that involves regulation of the proapoptotic forkhead transcription factors.

To understand how estrogen-responsive genes are regulated, we compared the abilities of estrogen receptors (ERs) alpha and beta to bind to and activate transcription through the consensus vitellogenin A2 ERE and the imperfect pS2, vitellogenin B1, and oxytocin (OT) EREs. Transient transfection experiments demonstrated that ERalpha and ERbeta induced the highest levels of transcription with the A2 ERE, intermediate levels of transcription with the OT ERE, and low levels of transcription with the pS2 and B1 EREs. ERalpha and ERbeta had higher affinities for the A2 ERE than for any of the three imperfect EREs but similar affinities for the pS2, B1, and OT EREs in gel mobility shift assays. ERalpha had a higher affinity and was a more potent activator of transcription than ERbeta. Interestingly, protease sensitivity assays demonstrated that A2, pS2, B1, and OT EREs induced distinct changes in ERalpha and ERbeta conformation thereby providing different functional surfaces for interaction with regulatory proteins involved in control of estrogen-responsive genes.

Antibodies are widely used to detect estrogen receptor (ER) in ER-DNA complexes in electrophoretic mobility shift assays (EMSA). We compared the specificity of antibodies raised to different regions of ERalpha or ERbeta for detecting recombinant human ERalpha (rhERalpha) and recombinant rat ERbeta (rrERbeta) when bound to a consensus estrogen response element (ERE). ERalpha-specific antibodies specifically slowed the migration of the ER-ERE complex by 32 to 84% and inhibited rhERalpha-ERE binding by 17 to 75%. None of antibodies to ERbeta supershifted rhERalpha-ERE complex. Some ERalpha-specific antibodies increased whereas some decreased rrERbeta-ERE binding. Anti-ERbeta antibodies supershifted different amounts of the rrERbeta-ERE complex. Our results indicate that supershift and inhibition of ER-ERE interaction with a specific antibody are equally reliable in the detection of rhERalpha and rrERbeta. ERalpha antibody Ab10, antisera G20 and AT3B, and ERbeta-antiserum Y19 offered the best discrimination between ERalpha and ERbeta. Comparison of the peptide sequences against which various antibodies were raised indicate directions for new ERalpha and ERbeta- specific antibody development. We conclude that a cognate ER antibody that retards the migration of the ER-ERE complex by at least 40% or inhibits ER-ERE interaction by at least 8% provides a reliable detection of a specific ER isoform in EMSA.

For approximately one-third of estrogen receptor (ER)-positive breast cancer patients, extracted tumor ER is unable to bind to its cognate DNA estrogen response element (ERE), an effect that is partly reversible by the thiol-reducing agent dithiothreitol (DTT). Full-length (67 kDa) ER or its 11 kDa recombinant DNA-binding domain (ER-DBD) is also susceptible to loss of structure and function by the action of oxidants such as diamide and hydrogen peroxide; however, prior DNA binding by ER or ER-DBD protects against this oxidant induced loss of function. The ER-DBD contains two (Cys)(4)-liganded zinc finger motifs that cooperate to stabilize a rigid DNA-binding recognition helix and a flexible helix-supported dimerization loop, respectively. Comparisons between synthetic peptide analogues of each zinc finger and recombinant ER-DBD in the presence of zinc by electrophoretic mobility shift assay, circular dichroism, and mass spectrometry confirm that cooperativity between these zinc fingers is required for both ER-DBD structure (alpha-helicity) and function (dimeric DNA binding). Rapid proteolytic digestion of monomeric, non-DNA-bound ER-DBD followed by HPLC-MS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNA-binding finger, but not the finger supporting the flexible dimerization loop, which remains sensitive to internal disulfide formation. These findings indicate that the loss of ER DNA-binding function in extracts from some primary breast tumors and in ER or ER-DBD exposed to thiol-reacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. Although ER-DBD contains several strategically located methionine residues, they are less susceptible to oxidation than the thiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNA-binding function.

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.

Molecular dynamics (MD) simulations of the estrogen receptor DNA-binding domain (ERDBD) as a dimer in complex with its DNA response element (ERE) show a significant difference in both structure and dynamics, compared to a MD simulation of monomeric ERDBD bound to its half-site response element (EREH). The C-terminal zinc binding domain (ZnII), including a region (helix II) which is in a helical conformation in ERE-(ERDBD)2, is considerably more flexible in EREH-ERDBD than in the dimeric complex. In EREH-ERDBD, all helical hydrogen bonds in helix II are broken and the entire ZnII region is detached from a hydrogen bonding network that in ERE-(ERDBD)2 connects to other parts of the protein as well as to the DNA. The regions that become flexible in EREH-ERDBD are identical to the regions where the NMR solution structure of free ERDBD is poorly ordered. This strongly suggests that dimerisation of ERDBD is required for ordering of the ZnII region and that monomeric binding to DNA is not sufficient for the ordering. This contrasts to the glucocorticoid receptor DNA-binding domain (GRDBD) which has essentially the same mobility (uniform and limited), regardless of whether it is free as a monomer in solution, bound as a monomer to its half-site response element or in a dimeric complex with the full response element. The hydrogen bonding network that connects ZnII with other parts of the protein and to DNA is almost identical in ERDBD and GRDBD. However, in GRDBD there is also a serine (in the N-terminal zinc coordinating region) with a central role in this network, connecting to the ZnII region. This serine is replaced by a glycine in ERDBD and we suggest that this substitution is sufficient for destabilisation of the network, thus leading to a more flexible ZnII region, which becomes ordered first upon forming a complex with another ERDBD and DNA.

Full-length (67 kDa) immunoreactive estrogen receptor (ER) extracted from a third of untreated ER-positive primary breast tumors appears unable to bind to its cognate estrogen response element (ERE). We have observed partial reversibility of this ER DNA-binding defect upon treatment of these tumor extracts with excess thiol reducing agent (DTT), suggesting that ER DNA-binding is subject to redox modulation as is reported for other zinc-finger proteins and transcriptional activators. Treatment of recombinant ER DNA-binding domain (ER-DBD) or ER-enriched extracts from CHO(ER) and MCF-7 cells with thiol-reacting oxidants (diamide, iodosobenzoate, H2O2) or alkylator (iodoacetamide) produces a dose-dependent loss in ER DNA-binding capacity. Thiol-specific oxidative loss in ER DNA-binding is fully reversible by DTT reduction, unlike the defect caused by thiol-specific alkylation. Circular dichroism spectrometry shows that both forms of treatment substantially modify ER secondary structure, inducing loss of alpha-helical content within the ER-DBD that is reversible after thiol oxidation but not after thiol alkylation. Oxidant (H2O2, menadione) exposure of cultured CHO(ER) or MCF-7 cells impairs the ability of endogenous ER to bind DNA and transactivate an ER-responsive reporter gene (ERE-tk-CAT), demonstrating that extracellular redox stress can modulate intracellular ER function. Since these thiol-specific oxidant and alkylator treatments have no significant effect on either recombinant ER ligand-binding or intracellular immunoreactive ER content, our findings suggest that DNA-binding and transactivation are the most sensitive intracellular ER functions impaired by oxidant stress in some ER-positive human breast tumors.

Estrogen receptor alpha was overexpressed in COS-7 cells by transformation of an expression vector with the full length open reading frame of the receptor alpha. The nuclear estradiol-receptor complex and the soluble receptor from the COS-7 cells were cross-linked with an estrogen response element (ERE), which was substituted with 5-bromo-2'-deoxyuridine (BrdUVRE), as the receptor dimers by UV irradiation. In gel retardation analysis, the specific bindings of both nuclear and soluble receptors to ERE were decreased with increasing of KCl concentration compared with 0.1 M KCl. The ionic interactions of both receptors with ERE are thought to be similar.

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes. ER-mediated transcriptional changes are brought about by interaction of the ER with the estrogen response element (ERE). In this study, we examined the interaction of the Xenopus laevis ER DNA binding domain (DBD) and the intact ER with the X. laevis vitellogenin A2 ERE and the human pS2 ERE. Using gel mobility shift, DNase I footprinting, and methylation interference assays, we demonstrated that the DBD bound only as a dimer to the A2 ERE. However, the DBD bound as a monomer to the consensus pS2 ERE half site at lower DBD concentrations and then as a homodimer to the consensus and imperfect pS2 ERE half site at higher DBD concentrations. Antibody supershift experiments carried out with partially purified, yeast-expressed full-length ER demonstrated that three ER-specific antibodies interacted differentially with A2 and pS2 ERE-bound ER, indicating that receptor epitopes were differentially exposed. Furthermore, partial digestion of the A2 and pS2 ERE-bound ER with chymotrypsin or trypsin produced distinct protease cleavage patterns. Taken together, these data provide evidence that differential interaction of the DBD with the A2 and pS2 EREs brings about global changes in ER conformation. The conformational changes in ER induced by individual ERE sequences could lead to association of the receptor with different transcription factors and assist in the differential modulation of estrogen-responsive genes in target cells.

Molecular dynamics simulations are carried out to investigate the binding of the estrogen receptor, a member of the nuclear hormone receptor family, to specific and non-specific DNA. Two systems have been simulated, each based on the crystallographic structure of a complex of a dimer of the estrogen receptor DNA binding domain with DNA. One structure includes the dimer and a consensus segment of DNA, ds(CCAGGTCACAGTGACCTGG); the other structure includes the dimer and a nonconsensus segment of DNA, ds(CCAGAACACAGTGACCTGG). The simulations involve an atomic model of the protein-DNA complex, counterions, and a sphere of explicit water with a radius of 45 A. The molecular dynamics package NAMD was used to obtain 100 ps of dynamics for each system with complete long-range electrostatic interactions. Analysis of the simulations revealed differences in the protein-DNA interactions for consensus and nonconsensus sequences, a bending and unwinding of the DNA, a slight rearrangement of several amino acid side chains, and inclusion of water molecules at the protein-DNA interface region. Our results indicate that binding specificity and stability is conferred by a network of direct and water mediated protein-DNA hydrogen bonds. For the consensus sequence, the network involves three water molecules, residues Glu-25, Lys-28, Lys-32, Arg-33, and bases of the DNA. The binding differs for the nonconsensus DNA sequence in which case the fluctuating network of hydrogen bonds allows water molecules to enter the protein-DNA interface. We conclude that water plays a role in furnishing DNA binding specificity to nuclear hormone receptors.

Unlike steroid and retinoid receptors, which associate with DNA as dimers, human estrogen related receptor-2 (hERR2) belongs to a growing subclass of nuclear hormone receptors that bind DNA with high affinity as monomers. A carboxyl-terminal extension (CTE) to the zinc-finger domain has been implicated to be responsible for determining the stoichiometry of binding by a nuclear receptor to its response element. To better understand the mechanism by which DNA specificity is achieved, the solution structure of the DNA-binding domain of hERR2 (residues 96-194) consisting of the two putative zinc fingers and the requisite 26-amino acid CTE was analyzed by multidimensional heteronuclear magnetic resonance spectroscopy. The highly conserved zinc-finger region (residues 103-168) has a fold similar to those reported for steroid and retinoid receptors, with two helices that originate from the carboxyl-terminal ends of the two zinc fingers and that pack together orthogonally, forming a hydrophobic core. The CTE element of hERR2 is unstructured and highly flexible, exhibiting nearly random coil chemical shifts, extreme sensitivity of the backbone amide protons to solvent presaturation, and reduced heteronuclear (1H-15N) nuclear Overhauser effect values. This is in contrast to the dimer-binding retinoid X and thyroid hormone receptors, where, in each case, a helix has been observed within the CTE. The implications of this property of the hERR2 CTE are discussed.

The interactions of estrogen receptor (ER) with monoclonal antibody (Mab) F9, developed against a synthetic 30-mer hybrid oligopeptide, were determined in the presence or absence of Mab NMT-1, raised against 15-mer peptide from the N-terminal A/B region (amino acids 140- 154) or Mab 213, raised to a peptide AT3 in the DNA-binding domain (amino acids 247-263). Mab F9 bound ER and formed a complex sedimenting at the approximately 11S region of the gradients. Mabs 213 and NMT-1 bound ER and formed complexes sedimenting at approximately 7S and 9S, respectively. Preincubation of ER with Mab 213, followed by reincubation with Mab F9, resulted in a complex sedimenting at the approximately 11S region of the gradients. Similarly, preincubation of ER with Mab NMT-1 followed by reincubation with Mab F9 also produced an approximately 11S complex on the gradients. These observations suggest that binding of Mab F9 to ER induced conformational changes causing the release of Mab 213 and Mab NMT-1 from ER. Furthermore, binding of Mab NMT-1 to the A/B region of ER also produced conformational changes causing the release of Mab 213 from its epitope in the DNA-binding region. These results indicate that binding of Mab F9 and Mab NMT-1, with epitopes located within amino acids 140-154 of the A/B region of ER, induced conformational changes in the DNA-binding domain, as determined by the inability of Mab 213 to remain bound to its epitope. These data further suggest that the DNA-binding region is sensitive to conformational changes induced in the native protein.

The estrogen receptor is a ligand-activated transcription factor that binds to specific DNA sequences, estrogen response elements. Recent studies have characterized the location of tyrosine and serine residues in the estrogen receptor that are phosphorylated either by purified protein kinases in vitro or in response to ligand and DNA binding in vivo. Here we examined how phosphorylation of purified bovine uterine estrogen receptor in vitro by casein kinase II impacts estrogen receptor-estrogen response element binding and 17 beta-estradiol ligand binding stability. Our results show that phosphorylation doubles estrogen receptor-estrogen response element binding, but does not affect estradiol binding stability. These finding suggest that phosphorylation by casein kinase II on serine residues within the A/B domain results in intramolecular interactions affecting the DNA binding domain but not the ligand binding domain of the estrogen receptor.

Tam-67 is an amino-terminal deletion mutant of c-Jun (delta3-122) lacking most of the c-Jun transactivation domain, which has been shown previously to function in a transdominant fashion to inhibit c-Jun-induced transactivation and cellular transformation. In order to create a ligand-dependent dominant negative repressor of AP-1, we have constructed a fusion of the TAM-67 gene with the ligand binding domain of the estrogen receptor. Fusion of TAM-67 with the ligand binding domain of the estrogen receptor produced a 68 kD protein (TAM-67ER) which was immunoprecipitated by c-Jun-specific and estrogen receptor-specific antisera and shown by gel retardation assay to bind oligonucleotides containing an AP-1 sequence. Cotransfection of TAM-67ER and an AP-1-dependent reporter construct into rat embryo cells demonstrated ligand specific inhibition of AP-1 transactivation. In the absence of hormone, TAM-67ER produced complete inhibition of c-Jun-induced AP-I transactivation. This inhibition was relieved by treatment with estradiol but not by treatment with tamoxifen. In addition, TAM-67ER inhibited activated c-Ha-ras- or c-raf-induced transformation of NIH3T3 cells. However, this inhibition of transformation was not relieved by the addition of estrogen. Thus, TAM-67ER inhibits transactivation in a ligand-dependent manner, but inhibits transformation in a ligand-independent manner. The results suggest that the ligand-dependent transactivation domain of the estrogen receptor (TAF-2) can substitute for the c-Jun transactivation domain absent in TAM-67 to stimulate transactivation. However, TAF-2 cannot substitute for the missing c-Jun transactivation domain to induce cellular transformation.

The kinetics of the interaction between recombinant human estrogen receptor and chicken vitellogenin gene II estrogen response element (ERE) were determined by ERE-Sepharose chromatography. The association constant of the interaction between the ERE and the human estrogen receptor was dependent on receptor concentration, estradiol binding and temperature. The highest association constant (80-100 x 10(6)M-1) was measured for the estradiol-bound receptor prepared at 25 degrees C and at concentrations higher than 7 nM. At high receptor concentrations (>7 nM) the binding mechanism of estradiol to the receptor was positive cooperative, indicating receptor homodimerization. At lower concentrations the binding mechanism was partially cooperative and the association constant of the liganded receptor was significantly lower. The binding mechanism at 4 degrees C was cooperative as well, and the association constants were similarly dependent upon receptor concentration, but were 50% lower than the receptor prepared at 25 degrees C. The association constant of the unliganded receptor was 4- to 5-fold lower than that of the liganded receptor at 25 degrees C. These data suggest that in addition to estradiol-induced conformational changes in the receptor, the receptor dimers are subjected to temperature-dependent changes, which further increase their affinity for an ERE.

We determined the amino acid and radiolabel sequences of tryptic [32P]phosphopeptides of the purified human estrogen receptor (hER) from MCF-7 cells and Sf9 cells. Serine 118 was identified as a site that was phosphorylated independently of estradiol-binding in MCF-7 cells. Proline is on the carboxy terminus of serine 118, which suggests that the serine-proline may be a consensus phosphorylation site motif for either the mitogen-activated protein (MAP) kinase or p34cdc2 kinase. MAP kinase selectively phosphorylated the recombinant hER in vitro on serine 118 independent of estradiol-binding, whereas p34cdc2 did not phosphorylate the hER. We demonstrated previously that serine 167 of the hER was phosphorylated in an estradiol-dependent manner. We therefore compared the consequence of hER phosphorylation at serine 118 by MAP kinase and phosphorylation at serine 167 by casein kinase II on the receptor's affinity for specific DNA binding. The binding of the hER to an estrogen response element was not altered by phosphorylation with MAP kinase at serine 118 but was significantly increased when phosphorylated at serine 167 by casein kinase II. These data suggest that phosphorylation of the hER by MAP kinase(s) pathways may influence receptor action by a mechanism other than the estradiol-dependent phosphorylation of hER by casein kinase II.

Molecular dynamics simulations and free energy perturbation calculations have been performed on the glucocorticoid receptor DNA-binding domain (GRDBD) in complex with three different half-site response elements (RE). These are the glucocorticoid receptor RE (GREH), the estrogen receptor RE (EREH) and an "intermediate" RE (GRE2H), where the base-pair 6A.T in GREH has been replaced by 6G.C. The molecular dynamics/free energy perturbation calculations reveal the same order of stability of the three complexes, i.e. GREH-GRDBD > GRE2H-GRDBD > EREH-GRDBD, as found experimentally, with reasonable quantitative agreement of the relative stabilities with the experiments. The more favourable entropy in associating GRDBD to GREH than to GRE2H could be explained by the additional non-polar surface that is removed from the solvent in the former complex formation. Part of this additional surface originates from the 6T methyl group in GREH. Sequence-dependent hydration patterns of the DNA major groove, which is interrupted by the GRDBD binding, could also contribute to the relatively more favourable entropy in forming GREH-GRDBD than GRE2H-GRDBD. The tenfold reduced cooperativity in the dimeric GRDBD binding to a partially palindromic ERE (palERE) compared to palGRE/GRE2 could be attributed to conformational changes in the residues of the "D-box" (Cys476 to Cys482) observed in the molecular dynamics simulations of EREH-GRDBD, but not in the other two complexes. Moreover, the conformations of the base-pairs at positions 5 and 6 are distorted (compared to free B-DNA) in GREH and GRE2H, due to the binding of GRDBD. Since the corresponding conformational distortions were not found in EREH-GRDBD, it is plausible that the distortions mediate cooperative binding through a propagation of the distortions to the other half site, thus facilitating binding of the second GRDBD.

The effect of estrogen structure on the conformation of the complex formed with estrogen receptor (ER) and the consensus estrogen response element (EREc) has been examined with gel mobility shift assay. Proteins in MCF-7 cell extracts formed three distinct complexes with ERE. Only the slowest moving complex contained ER as indicated by binding with anti-ER antibodies H222 and D547. This ER-ERE complex displayed increased electrophoretic mobility when formed in the presence of estradiol (E2) and bound radiolabeled 16 alpha-iodoestradiol. The antiestrogen ICI 164,384 decreased the mobility of the ER-ERE complex and blocked the effect of E2. The results reported here indicate that the position and location of hydroxyl groups on the estratriene nucleus is an important factor in determining the mobility of ER-EREc (or a variant ERE) in gel shift assays. The ability of E2 analogs to cause conformational changes detectable as altered mobility was not directly related either to their binding affinity for ER or to their ability to activate E2 responsive genes. Although several dihydroxyestrogens (estradiol-16 alpha, 1- and 2-hydroxyestratrien-17 beta-ol) caused an increase in the mobility of the ER-EREc, other ligands (estradiol-17 alpha, 4-hydroxyestratriene-17 beta-ol, 3-hydroxy estratriene, estratrien-17 beta-ol and 5-androsten-3 beta, 17 beta-diol) with a capacity for activating at least some E2 responsive genes in MCF-7 cells had little or no effect. On the basis of these and previously published results, it can be concluded that specific structure features of estrogens are responsible for conformational changes of ER-ERE complexes detectable in gel-shift assays. Furthermore, the identified structural characteristics of the ligand which are required for gel-shift are not the same as those previously reported to be essential for stimulation of transcriptional activity of ER.

Functional analyses, performed with the estrogen receptor (ER) isolated from different sources or produced with various expression systems, led to contradictory results concerning the role of estrogen (E2) and antiestrogens in ER DNA binding. Here we report the DNA-binding properties of the human ER and show that the wild type ER (HEG0) binds in vitro to an estrogen response element (ERE) as a dimer, irrespective of the presence or absence of estrogen. We also show that the two antihormones, 4-hydroxytamoxifen (OHT, a partial ER agonist) and ICI 164,384 (a pure antagonist) do not impair HEG0 dimerization and DNA binding in vitro. Exposure of HEG0 to elevated temperature (37 C) in vitro results in a much faster reduction of its binding capacity to an ERE in the absence of ligand or in the presence of ICI 164,384 than in the presence of either E2 or OHT. The Gly to Val mutation at amino acid 400 present in the human ER that we initially cloned (HE0), is responsible for an even faster heat inactivation of unliganded receptor compared with HEG0 and largely accounts for the previously observed in vitro ligand-dependent DNA binding of ER. We also show that, as previously observed for OHT, ICI 164,384 does not prevent ER binding to an ERE in vivo, even though ICI 164,384 acts as a pure antagonist for transcriptional activation by ER. We discuss these results in the context of a ligand-dependent interaction between the C-terminal region E, which contains the ligand-binding domain, and the N-terminal A/B region, which contains the activation function AF-1.

Steroid receptors recognize bipartite targets composed of six base-pair half-sites. There are two canonical types of half-site which differ only in their central two base pairs. The crystal structure of an estrogen receptor-like DNA-binding domain bound to the wrong type of half-site (a glucocorticoid response element) reveals an interface that resembles the specific interfaces of the glucocorticoid receptor or estrogen receptor bound to their correct response elements. The underlying stereochemical defect that weakens the non-cognate interface is a difference in the helical geometry of the incorrect DNA half-site which prevents a side-chain contact and results in a gap which is filled by at least five additional fixed water sites, imposing a potential entropic burden on the stability of the interface.

It has been suggested that cooperative binding of estrogen receptor (ER) may, in part, be responsible for the synergistic activation of transcription of estrogen-responsive genes that contain multiple estrogen-response elements (EREs). Experiments described here show that estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally occurring flanking sequences, such as an AT-rich region. In contrast, EREs lacking these sequences do not bind E2-ER cooperatively, regardless of ERE spacing or stereoalignment. Moreover, binding is of lower affinity and capacity in the absence of these critical flanking sequences. By varying the sequence of nucleotides adjacent to the ERE, features important for the flanking sequence effect were characterized. Interestingly, when ER was liganded with 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen, the antiestrogen-liganded ER complex (4-OHT-ER) did not bind cooperatively to multiple EREs, regardless of spacing or flanking sequence. We postulate that ERE flanking sequences bestow upon E2-ER enhanced ERE binding capacity and cooperativity, but do not affect 4-OHT-ER-ERE binding.

Background: Structural studies of protein-DNA complexes have tended to give the impression that DNA recognition requires a unique molecular interface. However, many proteins recognize DNA targets that differ from what is thought to be their ideal target sequence. The steroid hormone receptors illustrate this problem in recognition rather well, since consensus DNA targets are rare., Results: Here we describe the structure, at 2.6 A resolution, of a complex between a dimer of the DNA-binding domain from the human oestrogen receptor (ERDBD) and a non-consensus DNA target site in which there is a single base substitution in one half of the palindromic binding site. This substitution results in a 10-fold increase in the dissociation constant of the ERDBD-DNA complex. Comparison of this structure with a structure containing a consensus DNA-binding site determined previously, shows that recognition of the non-consensus sequence is achieved by the rearrangement of a lysine side chain so as to make an alternative base contact., Conclusions: This study suggests that proteins adapt to recognize different DNA sequences by rearranging side chains at the protein-DNA interface so as to form alternative patterns of intermolecular contacts.

The consensus oestrogen response element (ERE) contains two inverted copies of an AGGTCA consensus hexameric half-site, spaced by three base pairs. It differs from many other hormone response elements, such as consensus thyroid (TREp) and retinoic acid (DR-5 RARE) response elements, only in the relative spacing and orientation of these sequences. In the present study we report values for cooperativity (omega) of an oestrogen receptor DNA-binding domain polypeptide upon binding to these sequences. The polypeptide binds with negative cooperativity, or without cooperativity to retinoic acid and thyroid response elements respectively, but with high cooperativity to the ERE. We have also examined cooperativity upon binding of the polypeptide to an ERE variant. Since naturally occurring EREs commonly contain one hexamer which is considerably more degenerate than the other, we designed a hybrid response element in which one hexamer is a consensus ERE, while specific mutations were introduced into the other. We chose to mutate the second half-site to a glucocorticoid response element (GRE) half-site sequence (AGAACA), since normally no binding of the DNA-binding domain polypeptide to a GRE hexamer alone can be detected. In the hybrid response element, however, the GRE half-site is recognized with relatively high affinity, although binding to this sequence is dependent on the previous binding of a polypeptide to the ERE hexamer. Thus, cooperative interactions are capable of mediating the recognition of ERE sequence degeneracy. The ability of protein-protein interactions to mediate recognition of DNA sequence degeneracy may also have implications for transcription factors in general.

We have produced a truncated form of the human estrogen receptor (hER) as a fusion protein with glutathione S-transferase (GST) in Spodoptera frugiperda (Sf) cells using the baculovirus expression vector (BEV) system. The protein is correctly produced and can be purified from crude whole-cell extracts by a single-step, batch-wise affinity-purification procedure. We show that this GST-hER fusion protein binds at its DNA-binding site specifically and in a hormone-inducible manner. Furthermore, we used the purified hER to analyze the complex estrogen response element (ERE) in the promoter of the oxytocin-encoding gene.

To further our understanding of the role played by cysteine residues in ligand recognition by the human estrogen receptor (hER), we have individually mutated residues 381, 417, 447, and 530 within the hormone-binding domain from cysteine to serine. We have also examined 10 additional nonconservative amino acid changes at C530. Four mutants were identified with defects in their ability to stimulate transcription in response to hormone: C447S, C530P, C530W, and C530G. Accumulation of the mutant proteins to comparable steady state levels after transient expression in COS-7 cells leads us to conclude that none of these substitutions results in increased turnover of receptor protein. None of these mutations had a reduced affinity for estradiol when compared with wild-type hER [dissociation constant (Kd) = 0.29 nM]. Although C447 and C530 reside outside of motifs previously defined as being necessary for DNA binding or receptor dimerization, all four of these mutants unexpectedly displayed altered DNA-binding properties when analyzed using a gel mobility shift assay. Their loss of DNA binding could be overcome, at least in part, by hormone treatment or by the addition of antibody. Additionally, mutations C447S, C530S, and C530P displayed patterns of subcellular localization in the absence of hormone that differed from wild-type hER. These results argue that cysteine residues 447 and 530 within the hormone-binding domain play no direct role in the ability of this receptor to bind estradiol. These mutations, however, can effect DNA-binding activity and the distribution of hER within cells.

Two forms of nuclear estrogen receptors have been isolated and purified from the goat uterus. The biochemical characteristics of the proteins imply that the receptors may be identified as the type I and type II nuclear estrogen receptors. Nevertheless, we felt a necessity to exercise caution in using this nomenclature and, therefore, decided to identify them instead as R-I and R-II, respectively. While R-I is the DNA-binding form, R-II is a non-DNA-binding protein. The two proteins are totally dissimilar in other physical characteristics like the Stokes radii (36 A for R-I and 21 A for R-II), sedimentation coefficients (4.8 S for R-I and 3.8 S for R-II), the Kd (1 nM for R-I and 2 nM for R-II), and the nature of the CNBr fragmentation of the proteins. The two proteins, however, cross-react with polyclonal antibodies raised against goat uterine estrogen receptor activation factor (E-RAF), a DNA-binding protein with no capacity to bind estradiol, originally discovered by T.N.R.V. Thampan and J. H. Clark (1981, Nature 290, 152-154). A major feature of the R-II isolation procedure is the chromatography of the protein on a heat shock protein 90-Sepharose column in the presence of molybdate ions and elution using a molybdate-free buffer. While estradiol-17 beta (E2) binding to R-II was inhibited by the presence of dithiothreitol and quercetin in the medium, E2-R-I interaction remained unaffected by these exposures.

Background: The steroid/nuclear hormone receptors are a large family of conserved ligand-activated transcription factors that regulate gene expression through binding to response elements upstream of their target genes. Most members of this family bind to DNA as homodimers or heterodimers and recognize the sequence, spacing and orientation of the two half-sites of their response elements. The recognition and discrimination of the sequence and arrangements of these half-sites are mediated primarily by a highly conserved DNA-binding domain., Results: Here we describe the DNA-binding properties of the isolated DNA-binding domain of the oestrogen receptor, the ERDBD, and its refined NMR structure. This domain is monomeric in solution, but two molecules bind cooperatively to specific DNA sequences; this cooperativity determines the arrangement of half-sites that is recognized by the ERDBD. The 10 carboxy-terminal residues and a region of 15 residues within the domain are disordered in the solution structure, yet are important for DNA binding., Conclusion: The cooperative nature of ERDBD binding to DNA is important. The previously-determined X-ray structure of the ERDBD dimer bound to DNA shows that the 15 internal residues disordered in solution make contact both with DNA and with the corresponding region of the other monomer. These results suggest that these residues become ordered during the process of binding to DNA, forming the dimer interface and thus contributing to the cooperative interaction between monomers.

The nuclear hormone receptors are a superfamily of ligand-activated DNA-binding transcription factors. We have determined the crystal structure (at 2.4 A) of the fully specific complex between the DNA-binding domain from the estrogen receptor and DNA. The protein binds as a symmetrical dimer to its palindromic binding site consisting of two 6 bp consensus half sites with three intervening base pairs. This structure reveals how the protein recognizes its own half site sequence rather than that of the related glucocorticoid receptor, which differs by only two base pairs. Since all nuclear hormone receptors recognize one or the other of these two consensus half site sequences, this recognition mechanism applies generally to the whole receptor family.

The estrogen receptor (ER) is a strong hormone-inducible transcription factor that regulates the expression of many genes. It was shown for the human progesterone receptor that the binding of hormone causes distinct conformational changes in the ligand binding domain (LBD) and that these changes in LBD conformation are crucial for events after DNA binding. We now show that conformational changes in the LBD of the human ER are a prerequisite for trans-activation. Under the appropriate conditions ER binds to its response element and activates transcription only in the presence of ligand. Binding of the ligand causes changes in the conformation of the LBD. Antihormones induce distinct conformational changes, the differences between the conformations lying in the carboxy-terminal end of the receptor. Changing the experimental conditions results in a receptor that can bind to DNA and activate transcription in a ligand-independent manner. Under these conditions the LBD has a transcriptionally active conformation in the absence of ligand. Taken together, our data indicate that the conformational change induced by ligand is required for converting a receptor to the transcriptionally active form.

We present findings of genetic information conservation between the glucocorticoid response element (GRE) DNA and the cDNA encoding the glucocorticoid receptor (GR) DNA-binding domain (DBD). The regions of nucleotide sub-sequence similarity to the GRE in the GR DBD occur specifically at nucleotide sequences on the ends of exons 3,4, and 5 at their splice junction sites. These sequences encode the DNA recognition helix on exon 3, a beta-strand on exon 4, and a putative alpha-helix on exon 5, respectively. The nucleotide sequence of exon 5 that encodes the putative alpha-helix located on the carboxyl terminus of the GR DBD shares sequence similarity with the flanking nucleotide regions of the GRE. We generated a computer model of the GR DBD using atomic coordinates derived from nuclear magnetic resonance spectroscopy to which we attached the exon 5-encoded putative alpha-helix. We docked this GR DBD structure at the 39-base-pair nucleotide sequence containing the GRE binding site and flanking nucleotides, which contained conserved genetic information. We observed that amino acids of the DNA recognition helix, the beta-strand, and the putative alpha-helix are spatially aligned with trinucleotides identical to their cognate codons within the GRE and its flanking nucleotides.

The glucocorticoid receptor (GR) DNA binding domain consists of several conserved amino acids and folds into two zinc finger-like structures. Previous transactivation experiments indicated that three amino acids residing in this region, Gly, Ser and Val, appear to be critical for target-site discrimination. Based on the solved crystal structure, these residues are at the beginning of an amphipathic alpha-helix that interacts with the DNA's major groove; of these, only valine, however, contacts DNA. In order to examine their functional role directly, we have substituted these residues for the corresponding amino acids from the estrogen receptor (ER), overexpressed and purified the mutant proteins, and assayed their binding specificity and affinity by gel mobility shifts using glucocorticoid or estrogen response elements (GRE or ERE, respectively) as DNA probes. We find that all three residues are indeed required to fully switch GR's specificity to an ERE. The contacting valine in GR is of primary importance. The corresponding residue in ER, alanine, is less important for specificity, while glutamic acid, four amino acids towards the N-terminus, is most critical for ER discrimination. Finally, we show that the GR DNA binding domain carrying all three ER-specific mutations has a significantly higher affinity for an ERE than the ER DNA binding domain itself. We interpret these results in the context of both the data presented here and the crystal structure of the GR DNA binding domain complexed to a GRE.

Although the nucleotide bases that constitute the consensus DNA sequence of the estrogen response element (ERE) have been identified, the involvement of electrostatic contacts between the sugar-phosphate backbone of the ERE and the estrogen receptor (ER) is not known. Moreover, the contribution of these contacts to sequence-specific DNA binding has not been determined. Therefore, the interactions of highly purified ER with the phosphate residues of the ERE derived from the chick vitellogenin (cVit)-II gene were examined by phosphate ethylation interference. Specific ER-DNA complexes were evident in electrophoretic gel mobility shift assays using DNA fragments containing either the perfect ERE (-625 relative to gene start site; 5'-GGTCAGCGTGACC) or the imperfect ERE (-353; 5'-GGTCAACATAACC). The phosphate ethylation interference footprint identified a 2-fold, symmetrical exclusion of phosphate residues essential for specific binding to the perfect ERE with a 5' stagger, indicating that each monomer of the ER dimer is bound in the major groove of the DNA. The interference footprint of the imperfect ERE did not detect interactions between the receptor and the phosphate residues in the 3' half of the response element on the noncoding strand. In contrast, the corresponding footprint of the perfect ERE displayed strong interactions between the ER and the phosphate backbone of the DNA. Consequently, the absence of these electrostatic contacts very likely accounts for the reduced binding affinity of the ER for the imperfect ERE. These results indicate that specific contacts between the ER and the sugar-phosphate backbone of its cognate response elements are an important aspect of DNA sequence recognition and high affinity binding.

The affinity of the hormone-bound estrogen receptor for single-stranded and double-stranded DNA was compared using isocratic elution chromatography. The receptor bound single-stranded DNA with a two-fold higher affinity than double-stranded DNA (17.9 x 10(4) M-1 vs. 9.1 x 10(4) M-1) at 0.2 M KCl. The same number of ions were released when the receptor bound either single-stranded or double-stranded DNA (11.8 vs. 10.6, respectively). These results indicate the hormone-bound estrogen receptor has no strong preference for single-stranded vs. double-stranded nonspecific DNA, and has a similar conformation when bound to either form of DNA at physiological salt concentrations.