Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis - PubMed (original) (raw)

Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis

Alessandro Pandini et al. Biochemistry. 2007.

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that is activated by a structurally diverse array of synthetic and natural chemicals, including toxic halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Analysis of the molecular events occurring in the AhR ligand binding and activation processes requires structural information on the AhR Per-Arnt-Sim (PAS) B-containing ligand binding domain, for which no experimentally determined structure has been reported. With the availability of extensive structural information on homologous PAS-containing proteins, a reliable model of the mouse AhR PAS B domain was developed by comparative modeling techniques. The PAS domain structures of the functionally related hypoxia-inducible factor 2alpha (HIF-2alpha) and AhR nuclear translocator (ARNT) proteins, which exhibit the highest degree of sequence identity and similarity with AhR, were chosen to develop a two-template model. To confirm the features of the modeled domain, the effects of point mutations in selected residue positions on both TCDD binding to the AhR and TCDD-dependent transformation and DNA binding were analyzed. Mutagenesis and functional analysis results are consistent with the proposed model and confirm that the cavity modeled in the interior of the domain is indeed involved in ligand binding. Moreover, the physicochemical characteristics of some residues and of their mutants, along with the effects of mutagenesis on TCDD and DNA binding, also suggest some key features that are required for ligand binding and activation of mAhR at a molecular level, thus providing a framework for further studies.

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Figures

FIGURE 1

FIGURE 1

Cartoon representation of the PAS domain structures included in the analysis with their cofactors: HIF-2α (PDB ID 1P97), ARNT (PDB ID 1X0O), dPER (PDB ID 1WA9), HERG (PDB ID 1BYW), hPASK (PDB ID 1LL8), Phy3 (PDB ID 1G28), NCoA (PDB ID 1OJ5), FixL (PDB ID 1DRM), and PYP (PDB ID 1NWZ). Secondary structure attribution was according to the Kabsch and Sander method (58). For FixL and PYP the additional extra-domain elements included in the X-ray structures are also included (a long helix at the C-terminus for FixL and an N-terminal bundle of two helices for PYP).

FIGURE 2

FIGURE 2

(a) Diagram of the typical PAS fold with secondary structure elements labeled according to the nomenclature generally adopted for the PAS structures. (b) Sequence alignment of the mAhR against the templates HIF-2α and ARNT, pairwise aligned according to DALI. Only residues that are identical or similar for at least two of the three sequences are highlighted by colors. Coloring scheme for residues: red, acidic; blue, basic; purple, polar; yellow, Cys; brown, aromatic; green, hydrophobic; orange, Ser, Thr; gray, Pro, Gly. The mAhR predicted secondary structure and the template secondary structures, attributed according to the method of Kabsch and Sander (58), are also shown. Helices and β-strands are represented as white and black bars, respectively, and labeled with the PAS structure nomenclature (see panel a). Red arrows indicate the boundary residues of the mAhR cavity that have considerably smaller side chains than the corresponding ones in HIF-2α.

FIGURE 3

FIGURE 3

(a) Cartoon representation of the three modeled structures of the mAhR LBD. (b) Stick and cartoon representations of the model based on the HIF-2α and ARNT template structures (mod_HIF/ARNT) in different orientations, with the molecular surface (in blue) including the available volume in the cavity identified by CASTp. Secondary structure attribution was according to the method of Kabsch and Sander (58).

FIGURE 4

FIGURE 4

Cartoon representation of the modeled mAhR LBD (mod_HIF/ARNT) showing selected residues that were mutated. Residues with side chains pointing outside the modeled LBD are shown in blue; boundary residues of the cavity with side chains pointing inside it are shown in purple; Ile332, which is expected to have a structural role, is shown in yellow. The molecular surface (in green) including the available volume in the cavity identified by CASTp is shown.

FIGURE 5

FIGURE 5

Effect of mutation of selected residues within the mAhR LBD on TCDD-dependent AhR DNA binding. In vitro expressed wild-type or mutant AhR and wt ARNT were incubated with TCDD, and inducible AhR–ARNT–DRE complex formation was determined by gel retardation analysis as described under Materials and Methods. The positions of the induced AhR–ARNT–DRE complex are indicated by an arrow. Quantitation of the amount of the TCDD–AhR–ARNT–DRE complex was determined by phosphorimager analysis, and the results of multiple receptor preparations and gel retardation analyses (n ≥ 3) are presented in Table 4.

FIGURE 6

FIGURE 6

Expression levels of in vitro synthesized wild-type and mutant AhRs. 35S-Labeled wild-type and mutant AhRs were synthesized in vitro, denatured, and resolved by SDS–polyacrylamide gel electrophoresis and autoradiography of the dried gels as described in Materials and Methods. An arrow shows the bands of the AhR.

FIGURE 7

FIGURE 7

Stick representation of the helical connector main chain in the modeled mAhR LBD subjected to 1 ns MD simulation: (a) wild-type mAhR; (b) I332P mutant. The coloring scheme is according to the atom types. Hydrogen bonds are highlighted in yellow. Cartoon representation of the domain is shown in transparency.

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