In silico site-directed mutagenesis of the Daphnia magna ecdysone receptor identifies critical amino acids for species-specific and inter-species differences in agonist binding

摘要: Abstract Molting is an essential process in the life cycle of arthropods and regulated by complex neuroendocrine pathways where activation ecdysone receptor (EcR) plays a major role. The EcR forms non-covalent heterodimer with ultraspiracle protein (USP) when activated endogenous ecdysteroids, but can also be several insecticides other environmental chemicals. Environmental release exogenous chemicals may thus represent risk to non-target species due phylogenetic conservation arthropods. In present study, structural analysis homology models from freshwater crustacean Daphnia magna were used characterise agonist binding pocket identify amino acids responsible for differences between arthropod species. showed that pockets steroidal non-steroidal agonists are partly overlapping, phylogenetically conserved Thr59 key residue both types agonists. silico site-directed mutagenesis MM-GBSA dG calculations revealed Cys100 (D. numbering) determinant cross affinities. Other determinants Val129 agonists, Thr132 Asp134 results predict sensitivity shows modelling affinity predictions contribute identifying susceptible EcR-mediated endocrine disruption.