Comparative Analysis of Homology Models of the Ah Receptor Ligand Binding Domain: Verification of Structure–Function Predictions by Site-Directed Mutagenesis of a Nonfunctional Receptor

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity and response have been observed, the structural determinants responsible have not been determined and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of sixteen AHRs from twelve mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, His388) that reduce the internal space available to TCDD. Mutagenesis of two of these residues in zfAhR1a to those present in zfAHR2 (Y296H, T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue to examine species specific differences in AHR responsiveness.

Ligand Promiscuity of Aryl Hydrocarbon Receptor Agonists and Antagonists Revealed by Site-Directed Mutagenesis

“…6A and B). Both these amino acid residues are predicted to line the LBD cavity and are therefore termed fingerprint residues (33,60). In tern AhR, Val present in the position homologous to mouse Ile319 may contribute to the lower affinity of the tern AhR for TCDD (40).…”

Current basis for discovery and development of aryl hydrocarbon receptor antagonists for experimental and therapeutic use in atherosclerosis

“…For instance, the analysis of the Per-Arnt-Sim B subdomain from aryl hydrocarbon receptor nuclear translocator reveals that the highest degree of sequence identity is shared with the Per-Arnt-Sim B subdomain from aryl hydrocarbon receptor among all the Per-Arnt-Sim structures already reported (Pandini et al, 2007). When combined with site-directed mutagenesis, these theoretical models pointed out essential structural aspects from Per-Arnt-Sim B subdomain, thus guiding the identification of critical residues for the interaction of ligands inside the ligand binding domain as well as the activation of aryl hydrocarbon receptor (Fraccalvieri et al, 2013).…”

“…Per-Arnt-Sim B subdomain exhibits a buried and narrow pocket containing a volume of 300-600Ǻ 3 that preferentially accommodates planar ligands with a maximal dimension of 14Ǻx12Ǻx5Ǻ (Fraccalvieri et al, 2013). This pocket is surrounded and limited by Thr238, His285, Phe289, Pro291, Tyr316, Phe318, Ile319, His320, Cys327, Met334, Ala375 and Gln377.…”

“…Indeed, methoxy (MeO) or halogen substituents in aromatic chains dramatically increase the affinity for aryl hydrocarbon receptor, which ensures pure antagonist activity by reducing the dissociation rate and avoiding the associationdissociation dynamics that is critical to receptor activation during pharmacological agonism (de Medina et al, 2005). Finally, competitive antagonists would require short structures containing planar aromatic chains since the ligand binding domain pocket from aryl hydrocarbon receptor preferentially accommodates short planar ligands (Fraccalvieri et al, 2013).…”

Molecular Character, Phylogeny and Expression of Tomato LeNHX3 Gene Involved in Multiple Adverse Stress Responses

“…It has been previously reported that base substitutions can significantly alter gene function, for example, the Na + /H + antiporter SOS1 gene in Arabidopsis thaliana is essential for plant salt tolerance, however, a single base substitution in SOS1 made plants show salt-hypersensitive and low K + affinità [20,21], thus we speculate that the substituted amino acids in LeNHX3 may confer plants more pronounced salt tolerance. It has been demonstrated that homology model is accurate enough to predict protein structures in wide ranging applications [22,23], their folds are stabilized by inner residues contacts [24,25]. Kozachkov and Padan have reported that two residues at position 136 and 399 of Na + /H + antiporter NhaA in Escherichia coli were closely related to the conformational changes of protein [26].…”