Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.     

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

Ligand binding to ecdysone receptor (EcR) is an autonomous function of the ligand binding domain (LBD) and is not modified by other receptor domains or tags fused to the LBD. Association and dissociation velocity of hormone to EcR was studied in the absence and presence of its main dimerization partner Ultraspiracle (USP). Mutational analysis of the EcR(LBD) revealed that ligand entry and exit is affected differently by the same point mutation, indicating that different pathways are used for association and dissociation of the ligand. Heterodimerization with wild type USP(LBD) increases ligand association to EcR(LBD) about fivefold and reduces dissociation 18-fold. Opposite effects of the same mutation (N626K) on dissociation velocity of ligand in EcR and EcR/USP indicate that not only hormone binding itself, but also the kinetic behaviour of ligand binding is modified by the dimerization partner. A general effect of the point mutations on the 3D architecture seems unlikely due to the highly selective effects on the kinetics of hormone binding.     

Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster

Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP). The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding. Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648). Some amino acids are suboptimal for ligand binding (L615, T619), but mediate ligand-dependent dimerization. We conclude that the enhanced regulatory potential by ligand-dependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding. Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations. Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligand-binding pocket is modified by heterodimerization.     

Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle.

The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development.     

Functional characterization of ecdysone receptor gene switches in mammalian cells

Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).     

Heterodimerization of ecdysone receptor and ultraspiracle on symmetric and asymmetric response elements

Heterodimerization of nuclear receptors is facilitated by the interaction of two dimerization interfaces: one spanning the DNA-binding (C domain) region and the adjacent hinge (D domain) region, and the other in the ligand-binding (E domain) region. Ultraspiracle (USP) heterodimerizes with ecdysone receptor (EcR) and this complex participates in ecdysone signal transduction. The natural ecdysone response elements (EcREs) discovered so far are asymmetric elements composed of either imperfect palindromes or direct repeats. However, gel mobility shift assays have shown that both symmetric (perfect palindromes) and asymmetric (imperfect palindromes and direct repeats) elements can bind to the EcR/USP complex. Therefore, we analyzed EcR/USP domains involved in heterodimerization on different types of response elements (RE). Gel shift assays using full-length and truncated EcR and USP proteins showed that heterodimerization of these two proteins in the presence of asymmetric RE (DR4 and the natural EcRE hsp27) requires both dimerization interfaces present in CD and E domains of both proteins. In contrast, the dimerization interface present in the E domain of either EcR or USP was not essential for heterodimerization on symmetric RE such as PAL1 or IR1. We conclude that the use of heterodimerization interfaces present in CD and E domains of EcR/USP depends on the nature of response elements they bind to.     

Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors

Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.     

Critical role of desolvation in the binding of 20-hydroxyecdysone to the ecdysone receptor

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

Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects

Cloned EcR and USP cDNAs encoding the ecdysone receptors of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were manipulated to allow the co-expression of their ligand binding domains (LBDs) in insect cells using a baculovirus vector. Recombinant DE/F segment pairs (and additionally, for H. armigera, an E/F segment pair) from the EcR and USP proteins associated spontaneously with high affinity to form heterodimers that avidly bound an ecdysteroid ligand. This shows that neither ligand nor D-regions are essential for the formation of tightly associated and functional LBD heterodimers. Expression levels ranged up to 16.6mg of functional apo-LBD (i.e., unliganded LBD) heterodimer per liter of recombinant insect cell culture. Each recombinant heterodimer was affinity-purified via an oligo-histidine tag at the N-terminus of the EcR subunit, and could be purified further by ion exchange and/or gel filtration chromatography. The apo-LBD heterodimers appeared to be more easily inactivated than their ligand-containing counterparts: after purification, populations of the former were <40% active, whereas for the latter >70% could be obtained as the ligand-LBD heterodimer complex. Interestingly, we found that the amount of ligand bound by recombinant LBD heterodimer preparations could be enhanced by the non-denaturing detergent CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate). Purity, integrity, size and charge data are reported for the recombinant proteins under native and denaturing conditions. Certain intra- and intermolecular disulfide bonds were observed to form in the absence of reducing agents, and thiol-specific alkylation was shown to suppress this phenomenon but to introduce microheterogeneity.     

昆虫蜕皮激素受体及其类似物的杀虫机制研究进展

昆虫的蜕皮、变态和繁殖受到蜕皮激素的严格调控。蜕皮激素作用靶标由蜕皮激素受体（ecdysteroid receptor, EcR）和超气门蛋白（ultraspiracle protein, USP）组成,蜕皮激素与EcR/USP作用启动蜕皮级联反应过程。昆虫EcR具有种类或类群的特异性,研究其结构、功能和调控机理在开发环境友好型新药剂和基因调控开关等方面具有重要指导作用。该文介绍了昆虫EcR的结构和功能特点,蜕皮激素及其类似物与EcR/USP的分子作用方式,以及基于EcR/USP的新杀虫剂创制和基因调控开关设计等方面的重要进展。     

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of beta-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.     

Ligand binding by recombinant domains from insect ecdysone receptors

The ligand binding domains (LBDs) from the EcR and USP proteins of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were purified as recombinant heterodimers. The K(d) values for [(3)H]-ponasterone A binding by LBD heterodimers that included the hinge regions (i.e., DE/F heterodimers) ranged 0.7-2.5 nM, with K(i) values for ecdysteroid and dibenzoylhydrazine ligands ranging from 0.1 nM to >448 microM. The K(d) and K(i) values for a recombinant H. armigera LBD heterodimer that lacked D-regions (i.e., an E/F heterodimer) were approximately 4 times higher than those for its DE/F counterpart. Rate constants were estimated for the L. cuprina LBD heterodimer. A fluorescein-inokosterone conjugate (K(i)~40 nM) was used to develop a novel binding assay based on fluorescence polarization. This assay, which ranked the affinity of competitor ecdysteroids in the same order as the [(3)H]-ponasterone A binding assay, is well suited to high-throughput screening. Ponasterone A had a higher affinity than muristerone A for the recombinant hemipteran LBD heterodimers, whereas the reverse was true for the recombinant dipteran one. The same preference was observed when these ligands were tested as inducers of ecdysone receptor-controlled gene expression in transfected mammalian cells. The binding data obtained in vitro using recombinant LBD heterodimers reflects the ability of agonists to induce transgene expression in recombinant mammalian cells, and can also reflect their efficacy as larvicides.     

Mutations in the “zinc fingers” or in the n-terminal region of the DNA binding domain of the human glucocorticosteroid receptor facilitate its salt-induced transformation, but do not modify hormone binding

While the effects of the ligand (hormone)_binding domain (LBD) on other receptor domain functions are kwown, the effects of other domains on LBD functions have not been studied. In this work, we examined the importance of the strutural integrity of other domains of the human glucocorticosteroid receptor (hGR) on LBD activity (stability of 8S complexes, binding of hormone, and transformation from the 8S to the 4S form). Several mutations introduced outside the LBD affect neither the formation of stable 8S heterooligomeric complexes nor the hGR binding affinity for the agonist triacinolone acetonide (TA) or the 8S-hGR into a 4S form. Deletion of the second zinc finger of the DNA binding domain (DBD) facilitated 8S dissociation whether the ligand was TA or RU486. Deletion of the first zinc finger facilitated dissociation only in the presence of RU486. Deletion of the first zinc finger facilitated dissociation only in the presence of RU486. while replacement of PRO 416 (in the N-terminal region of the DBD) by ARG destabilized the 8S form only in the presence of TA. Variations in the salt-sensitivity of the mutated 8S GR complexes as a function of the ligand suggest that the DBD may interact functioanlly (if not physically) with the LBD. This interaction (possibly mediated by hsp90) could be influenced by minor structural differences between agonist and antagonist-GR complexes.     

Exploring the binding site structure of the PPAR gamma ligand-binding domain by computational solvent mapping

Solvent mapping moves molecular probes, small organic molecules containing various functional groups, around the protein surface, finds favorable positions, clusters the conformations, and ranks the clusters based on the average free energy. Using at least six different solvents as probes, the probes cluster in major pockets of the functional site, providing detailed and reliable information on the amino acid residues that are important for ligand binding. Solvent mapping was applied to 12 structures of the peroxisome proliferator activated receptor gamma (PPARgamma) ligand-binding domain (LBD), including 2 structures without a ligand, 2 structures with a partial agonist, and 8 structures with a PPAR agonist bound. The analysis revealed 10 binding "hot spots", 4 in the ligand-binding pocket, 2 in the coactivator-binding region, 1 in the dimerization domain, 2 around the ligand entrance site, and 1 minor site without a known function. Mapping is a major source of information on the role and cooperativity of these sites. It shows that large portions of the ligand-binding site are already formed in the PPARgamma apostructure, but an important pocket near the AF-2 transactivation domain becomes accessible only in structures that are cocrystallized with strong agonists. Conformational changes were seen in several other sites, including one involved in the stabilization of the LBD and two others at the region of the coactivator binding. The number of probe clusters retained by these sites depends on the properties of the bound agonist, providing information on the origin of correlations between ligand and coactivator binding.