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.     

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.     

1H NMR studies of DNA recognition by the glucocorticoid receptor: complex of the DNA binding domain with a half-site response element.

The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by H?rd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505].     

Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element

Gel retardation analysis with full- and half-palindromic sequences using partially purified glucocorticoid receptor (GR) resulted in GR-glucocorticoid response element (GRE) species of identical mobilities, suggesting that formation of the dimeric GR protein complex is not catalyzed by DNA binding. These results are in contrast to the behavior of the isolated DNA binding domain of the glucocorticoid receptor where dimerization occurred on the GRE. Density gradient centrifugation of cytosolic GR resulted in two forms, a 4 S peak characteristic of the monomeric GR and a fraction which sediments at 6 S which is consistent with the observed size of the dimeric GR. These two forms were found to differ in their ability to bind to specific DNA sequences with the 6 S species having a higher affinity for a GRE. Taken together our results are consistent with a two-step model for hormone-induced transformation of GR: dissociation of the multimeric untransformed complex and dimerization of the GR to yield a high affinity DNA binding species.     

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.     

Ligand binding is without effect on complex formation of the ligand binding domain of the ecdysone receptor (EcR)

The ligand-binding domain (LBD) encompassing the C-terminal parts of the D- and the complete E-domains of the ecdysteroid receptor (EcR) fused to Gal4(AD) is present in two high molecular weight complexes (600 and 150 kDa) in yeast extracts according to size exclusion chromatography (Superdex 200 HR 10/30). Hormone binding is mainly associated with 150-kDa complexes. Complex formation is not influenced by hormone, but the ligand stabilizes the complexes at elevated salt concentrations. Mutational analysis of Gal4(AD)-EcR(LBD) revealed that formation of 600-kDa, but not 150-kDa, complexes depends on dimerization mediated by the EcR(LBD). Deletion of helix 12 is without effect. Mutation of K497 in helix 4, known to be essential for comodulator binding, abolishes 600-KDa complexes, but does not interfere with the formation of 150-kDa complexes. In contrast, the DE-domains of USP fused to Gal4(DBD) elute as monomer after elimination of the dimerization capacity of the ligand-binding domains by mutation of P463 in helix 10. The data presented here reveal that the complex formation of ligand-binding domains EcR and USP ligand is different.     

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.     

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending.

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.     

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.     

DNA binding and dimerization determinants for thyroid hormone receptor alpha and its interaction with a nuclear protein.

The gel retardation assay was used to analyze the role of the thyroid hormone receptor alpha (TR alpha) ligand-binding domain (LBD) in controlling receptor interaction with a thyroid hormone responsive element (TRE). While wild type receptor TR alpha binds to the TRE mainly as monomer, deletion of 85 amino acids from its C-terminus results in a mutant receptor with enhanced DNA binding that forms several slow mobility complexes as revealed by gel retardation assay. Receptor deletion mutants that lack most of the LBD show significantly elevated DNA binding and are still able to bind to DNA as two complexes. Thus, the C-terminal end of TR alpha appears to interfere with the dimerization/oligomerization function and DNA binding of TR alpha. All C-terminal deletion mutants have lost their T3-responsive activator function, but some show constitutive activity. Nuclear factor from several cell lines, including CV-1, F9, and GC cells, interacts with TR alpha receptor to form a larger molecular weight complex as determined by gel retardation assay. This factor could not be detected in HeLatk- cells, where TR alpha does not activate a TRE-containing reporter gene. The nuclear factor is heat sensitive and does not bind to TRE itself but can interact with TR alpha in the absence of DNA. Deletion analysis demonstrates that the leucine zipper-like sequence located in the LBD of TR alpha is involved in this interaction. Together, our data suggest that TR alpha contains a dimerization function outside the LBD which is inhibited by the carboxy-terminal region, while the leucine zipper-like sequence in the LBD is required for interaction with a nuclear factor.     

Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction.

HIV-1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C-terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence-specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction.     

Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding

NHP6A is a chromatin-associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three alpha-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C,15N-labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.     

Stereochemical aspects of interaction of DNA binding domain of human progesterone receptor with d(AGGTCATGCT)2.

Structures of (i) 66 amino-acid fragment (residues 567-633) from DNA binding domain of human progesterone receptor (hPR), (ii) a ten base pair DNA sequence d(AGGTCATGCT)2 from hormone responsive element (HRE) and (iii) a complex of these two are optimised by computer modelling and molecular mechanics technique using extensive steric constraints from secondary structure predictions, comparison with the structures of known metalloproteins, geometric constraints imposed by tetrahedral coordination with the zinc ion and comparison with structures of DNA binding domains of human glucocorticoid and estrogen receptors (hGR and hER). Structure of the complex was obtained using genetic modification data on steroid receptors and general consensus about protein-DNA interaction. DNA is in distorted B conformation. Sequence dependent as well as protein-induced conformation changes are noticed. There is change in propeller twist, buckle and angle between glycosyl bonds. However, H-bonding network is preserved. The complex is stabilized with eighteen hydrogen-bonds, mainly between peptide side-chains and backbone phosphate. There are five specific H-bonds between basic amino acid side chains, Lys 22, Lys 26 and Arg 27, and DNA bases, A1, G3, G16 and A17. Gly 19, Ser 20 and Val 23 are in close proximity of DNA.     

Specific binding of estrogen receptor to the estrogen response element.

Gene transfer studies have shown that estrogen regulation of specific genes is mediated by estrogen response elements (ERE). We report that binding of the estrogen receptor to the ERE can be detected by a gel retardation (band shift) assay. This binding interaction was highly sequence and receptor specific. Methylation interference analysis showed that the ERE contact sites of estrogen receptor displayed a perfect twofold rotational symmetry. This is compatible with estrogen receptor binding to the ERE as a head-to-head dimer.     

Structure-function studies of DNA binding domain of response regulator KdpE reveals equal affinity interactions at DNA half-sites

Expression of KdpFABC, a K⁺ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABC_BS) via the winged helix-turn-helix type DNA binding domain (KdpE_DBD). Exploration of E. coli KdpE_DBD and kdpFABC_BS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpE_DBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpE_DBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpE_DBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins.