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 for those differences 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 16 AHRs from 12 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 those of 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, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and 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 for examining species-specific differences in AHR responsiveness.

FIGURE 1

Cartoon representation of the homology models of three AHR LBDs with high affinity for TCDD, representative in each class. In the upper part, mouse, chicken and zebrafish AHR (mAHR, chAHR, zfAHR2) cartoons are colored according to the Secondary Structure attribution obtained by DSSPcont (45) (red: helices, yellow: β-strands). The secondary structure elements of the mAHR are labeled according to the nomenclature generally adopted for the PAS structures. In the lower part, cartoons are colored in grey, and the molecular surfaces that include the consensus cavity of the high affinity mammalian AHRs (cyan), the chAHR (green) and the zfAHR2 (orange) cavities are shown. Representation of the cavity surfaces was produced with PyMOL (47).

FIGURE 2

Ligand binding cavities of mammalian and avian AHRs. A. Sequence alignment of selected mammalian and avian AHRs. Except for the mAHR (taken as reference), only the residues identified by the CastP server (48) as internal to the binding cavities are shown. Internal residues conserved in all mammalian AHRs with high affinity for TCDD are indicated by asterisks. B. Cartoon representation of the modeled mAHR LBD. The internal residues conserved in all high affinity mammalian AHRs are shown as grey sticks and the residues that are not conserved in mammalian and avian AHRs with medium or low TCDD affinity are labeled and shown as red sticks. The molecular surface of the consensus cavity for mammalian AHRs is shown in cyan. C. Stick representation of the mAHR (cyan), mDBAAHR (yellow) and huAHR (pink) residues in the “TCDD binding fingerprint” positions (18). Steric hindrance of unconserved residues is shown as van der Waals spheres around the side chains. D. Stick representation of the residues of the avian AHRs (chAHR in green and tAHR in magenta) in the TCDD binding fingerprint positions, compared to those of mAHR (cyan). Van der Waals spheres are shown around the side chains of unconserved residues.

FIGURE 3

Ligand binding cavities of fish AHRs. A. Sequence alignment of zfAHR2, zfAHR1b and zfAHR1a. Except for the zfAHR2 (taken as reference), only the residues identified by the CastP server (48) as internal to the binding cavities are shown. B. Cartoon representation of the modeled zfAHR2 LBD. The internal residues are shown as grey sticks and the residues that are different in the zfAHR1a (lacks TCDD binding) are labeled and shown as black sticks. The molecular surface of the internal cavity is shown in orange. C. Cartoon representation of the modeled zfAHR1b LBD. The internal residues are shown as grey sticks and the residues that are different in zfAHR1a are labeled and shown as black sticks. The molecular surface of the internal cavity is shown in red. D. Cartoon representation of the modeled zfAHR1a LBD. The internal residues are shown as grey sticks and residues that are different with respect to the zfAHRs that bind TCDD are labeled and shown as black sticks. The molecular surface of the truncated zfAHR1a cavity is shown in blue. E. The steric hindrance of Tyr296, Thr386 and His388 of zfAHR1a (shown as blue Van der Waals spheres) results in truncated internal cavity (blue). The corresponding residues in the high affinity TCDD-binding zfAHR2 are shown in orange.

FIGURE 4

[³H]TCDD specific binding to wild-type zebrafish AHR1a, AHR1b and AHR1a containing various mutations. Wild-type and mutant zfAHRs were synthesized in vitro and subjected to [³H]TCDD ligand binding analysis by sucrose density centrifugation as described under Methods. Specific binding is expressed as a percentage of the specific binding to AHR1b, measured in the same experiment. Top panel shows results compiled from three experiments, with 2 or 3 replicate samples analyzed for each AHR. Bottom panels show representative results from one of the experiments in which all AHRs were analyzed. Note the difference in scale on the two panels.

FIGURE 5

Effect of site-directed mutagenesis of AHR1a on its ability to transform and bind to DNA in a TCDD-inducible manner. A. Wild-type zfAHR2, zfAHR1b, and zfAHR1a containing various mutations were synthesized in vitro, incubated in the presence of DMSO (D) or 20 nM TCDD (T) and transformation and DNA binding assessed by gel retardation analysis as described under Methods. B. Constitutive and inducible protein-DNA complexes in the dried gel were quantitated using a Fujifilm FLA9000 imager with Multi Gauge software and values represent the mean ± SD of triplicate binding reactions. A typical gel retardation analysis is shown.

FIGURE 6

Effect of site-directed mutagenesis of AHR1a on its ability to activate transcription. COS-7 cells were transfected with expression constructs for the zebrafish ARNT2b (25 ng), pGudLuc6.1 (20 ng), pGL4.74 (transfection control), and the indicated AHR expression constructs (5 ng). “No AHR” indicates transfection with only the reporter construct and the transfection control. The cells were exposed to dimethyl sulfoxide or TCDD (10 nM). The luciferase activity was measured in a luminometer and the relative luciferase units were calculated by normalizing the firefly luciferase activity to the transfection control Renilla luciferase.