title: Specific ligand binding domain residues confer low dioxin responsiveness to AHR1β of Xenopus laevis.
abstract: The aryl hydrocarbon receptor (AHR) is a Per-ARNT-Sim (PAS) family protein that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vertebrates. Frogs are remarkably insensitive to TCDD, and AHRs from Xenopus laevis bind TCDD with low affinity. We sought to identify structural features of X. laevis AHR1β associated with low TCDD sensitivity. Substitution of the entire ligand binding domain (LBD) with the corresponding sequence from mouse AHR(b-1) dramatically increased TCDD responsiveness in transactivation assays. To identify the amino acid residues responsible, we constructed a comparative model of the AHR1β LBD using homologous domains of PAS proteins HIF2α and ARNT. The model revealed an internal cavity with dimensions similar to those of the putative binding cavity of mouse AHR(b-1), suggesting the importance of side chain interactions over cavity size. Of residues with side chains clearly pointing into the cavity, only two differed from the mouse sequence. When A354, located within a conserved β-strand, was changed to serine, the corresponding mouse residue, the EC50 for TCDD decreased more than 15-fold. When N325 was changed to serine, the EC50 decreased 3-fold. When the mutations were combined, the EC50 decreased from 18.6 to 0.8 nM, the value nearly matching the TCDD sensitivity of mouse AHR. Velocity sedimentation analysis confirmed that mutant frog AHRs exhibited correspondingly increased levels of TCDD binding. We also assayed mutant AHRs for responsiveness to a candidate endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ). Mutations that increased sensitivity to TCDD also increased sensitivity to FICZ. This comparative study represents a novel approach to discerning fundamental information about the structure of AHR and its interactions with biologically important agonists.
authors: Biology Department, Kenyon College , Gambier, Ohio 43022, United States.
authors: Camila
authors: C
authors: Odio
authors: Sarah A
authors: SA
authors: Holzman
authors: Michael S
authors: MS
authors: Denison
authors: Domenico
authors: D
authors: Fraccalvieri
authors: Laura
authors: L
authors: Bonati
authors: Diana G
authors: DG
authors: Franks
authors: Mark E
authors: ME
authors: Hahn
authors: Wade H
authors: WH
authors: Powell
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002227:Carbazoles / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism / Q000633:toxicity*; D017434:Protein Structure, Tertiary; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015533:Transcriptional Activation; D029867:Xenopus Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D002227:Carbazoles; D008024:Ligands; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C111855:6-formylindolo(3,2-b)carbazole; C484722:Ahr protein, mouse; C476769:aryl hydrocarbon receptor, Xenopus
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