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 AHRb-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 AHRb-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.

Odio, C., Holzman, S., Denison, M., Fraccalvieri, D., Bonati, L., Franks, D., et al. (2013). Specific Ligand Binding Domain Residues Confer Low Dioxin Responsiveness to AHR1β of Xenopus laevis. BIOCHEMISTRY, 52(10), 1746-1754 [10.1021/bi301722k].

Specific Ligand Binding Domain Residues Confer Low Dioxin Responsiveness to AHR1β of Xenopus laevis

FRACCALVIERI, DOMENICO;BONATI, LAURA;
2013

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 AHRb-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 AHRb-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.
Articolo in rivista - Articolo scientifico
AHR, LBD, Homology Models, TCDD
English
2013
52
10
1746
1754
none
Odio, C., Holzman, S., Denison, M., Fraccalvieri, D., Bonati, L., Franks, D., et al. (2013). Specific Ligand Binding Domain Residues Confer Low Dioxin Responsiveness to AHR1β of Xenopus laevis. BIOCHEMISTRY, 52(10), 1746-1754 [10.1021/bi301722k].
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/42074
Citazioni
  • Scopus 20
  • ???jsp.display-item.citation.isi??? 17
Social impact