Structural adaptability in the ligand-binding pocket of the ecdysone hormone receptor. Nature 426:91-96

Département de Biologie et de Génomique Structurales, IGBMC, CNRS/INSERM/Université Louis Pasteur, Parc d'Innovation BP10142, 67404 Illkirch cedex, France.
Nature (Impact Factor: 41.46). 12/2003; 426(6962):91-6. DOI: 10.1038/nature02112
Source: PubMed


The ecdysteroid hormones coordinate the major stages of insect development, notably moulting and metamorphosis, by binding to the ecdysone receptor (EcR); a ligand-inducible nuclear transcription factor. To bind either ligand or DNA, EcR must form a heterodimer with ultraspiracle (USP), the homologue of retinoid-X receptor. Here we report the crystal structures of the ligand-binding domains of the moth Heliothis virescens EcR-USP heterodimer in complex with the ecdysteroid ponasterone A and with a non-steroidal, lepidopteran-specific agonist BYI06830 used in agrochemical pest control. The two structures of EcR-USP emphasize the universality of heterodimerization as a general mechanism common to both vertebrates and invertebrates. Comparison of the EcR structures in complex with steroidal and non-steroidal ligands reveals radically different and only partially overlapping ligand-binding pockets that could not be predicted by molecular modelling and docking studies. These findings offer new perspectives for the design of insect-specific, environmentally safe insecticides. The concept of a ligand-dependent binding pocket in EcR provides an insight into the moulding of nuclear receptors to their ligand, and has potential applications for human nuclear receptors.

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Available from: Natacha Rochel, Oct 25, 2015
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    • " 2007 ) . Therefore , there is a possibility that the ligand can bind to a pre - ferred conformer , and conversely , the ligand can induce conformation changes and the geometry of the binding site after binding . In fact , many examples of such ligand - induced plasticity of active sites are known . These include the Drosophila ecdysone receptor ( Billas et al . , 2003 ) , pregnane X receptor ( Chrencik et al . , 2005 ) , liver X receptor ( Farnegardh et al . , 2003 ) , peroxisome prolif - erator - activated receptor ( Xu et al . , 2002 ) , urinary protein MUP - 1 receptor ( Timm , Baker , Mueller , Zidek & Nov - otny , 2001 ) , glucocorticoid receptor ( Suino - Powell et al . , 2008 )"
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    ABSTRACT: C1 domains are small zinc-binding structural units of approximately 50 amino acids, originally discovered as lipid-binding modules in protein kinase C (PKC) isoforms. C1 domains that bind and respond to the DAG/phorbol ester are termed as typical, and those that do not respond to DAG/phorbol ester are termed as atypical. To design molecules targeting a specific C1 domain for regulating a specific disease state, it is important to understand the factors that make a C1 domain responsive to DAG/phorbol ester. Here, we determined the volume and surface area of the ligand-binding site for all known C1 domains. No correlation was found between the volume/surface area of ligand-binding site and the DAG/phorbol ester-binding affinity. Solvated molecular dynamics simulation reveals that the presence of water molecules affects the flexibility of the ligand-binding site. Contributions of the binding site residues, their orientations, and the membrane lipids on the responsiveness of a C1 domain towards DAG/phorbol ester have been discussed.
    Full-text · Article · Mar 2014 · Journal of biomolecular Structure & Dynamics
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    • "The DNA-binding domain (DBD), also called the C-domain, and the ligand-binding domain (LBD) or E-domain are the most conserved across all taxa for both receptors. A ligand-binding pocket, which has been characterized by X-ray crystallography (Billas et al., 2003; Carmichael et al., 2005), is formed within the E-domain of EcR heterodimerized to USP-RXR. The D-domain, also referred to as the hinge region, laying between the C-and E-domains, can have an influence on ligand binding as seen in some insect EcRs (Graham et al., 2007). "
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    ABSTRACT: In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. Major results were: (i) cDNAs encoding the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likelihood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacostracan clade (bootstrap percentage=75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the common shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein-protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100μg/L, and no effects against the molting process and nymphal development were scored. In conclusion, the in vitro cell reporter assay, based on NiEcR-LBD/CrcRXR-LBD heterodimerization in Hi5 cells and validated with the natural ecdysteroid hormone PonA, represents a useful tool for the screening of putative EDCs. As a test example for non-steroidal ecdysone agonist insecticides, tebufenozide had no negative effects on NiEcR/RXR receptor dimerization in vitro, nor on the molting process and nymphal development of N. integer at the tested concentration (100μg/L) in vivo.
    Full-text · Article · Jan 2013 · Aquatic toxicology (Amsterdam, Netherlands)
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    • "For EcR, the LBD plays roles in receptor dimerization, ligand recognition and cofactor interactions. Recently, the flexible ligand-dependent binding pocket where steroidal and non-steroidal binds, has been characterized by X-ray crystallography [10-13]. The F domain is divergent and its functions are still unknown. "
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    ABSTRACT: Background The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. Result In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM). In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. Conclusions Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists) to their targets (ecdysone receptors) leads to an adaptive response (resistance), is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.
    Full-text · Article · Oct 2012 · BMC Molecular Biology
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