Structural and Functional Characterization of the Aryl Hydrocarbon Receptor Ligand Binding Domain by Homology Modeling and Mutational Analysis

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,. 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 2α (HIF-2α) 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.The aryl hydrocarbon receptor (AhR) 1 is a basic helix-loop-helix (bHLH), PAS-(Per-ArntSim-) containing transcription factor which is present in numerous species and tissues and activates gene expression in a ligand-dependent manner (1-3). While the AhR can bind and be activated by a large number of structurally diverse chemicals (4-6), the highest affinity ligands include halogenated aromatic hydrocarbons (HAHs), such as 2, 3,7,8- NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 28. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and polycyclic aromatic hydrocarbons (PAHs), both widespread classes of environmental contaminants (4,7). Mechanistically, the inducing chemical diffuses across the plasma membrane and binds to the cytosolic AhR which exists as a multiprotein complex containing two molecules of hsp90 (a heat shock protein of 90 kDa), the X-associated protein 2 (XAP2), and the cochaperone p23 (8).Following ligand binding, the AhR is presumed to undergo a conformational change (9), exposing an N-terminal nuclear localization sequence that leads to translocation of the liganded AhR complex into the nucleus (10). Dissociation of the AhR from the protein complex and its dimerization with ARNT (AhR nuclear translocator) convert the AhR complex into its high-affinity ...

Docking and 3D-QSAR studies on the Ah receptor binding affinities of polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs)

“…2. The results are consistent with the findings of relevant reports (Procopio et al, 2002;Pandini et al, 2007). Therefore, the modeled AhR could be used for the following mechanism exploration.…”

“…The LBD of modeled AhR contained five ␤-sheets and one ␣-helix, which was in accordance with previous investigations (Denison et al, 2002;Pandini et al, 2007). A Ramachandran plot from PROCHECK ( Fig.…”

Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor

“…An example of this is the PAS-B domain of the aryl hydrocarbon receptor (AHR), which binds a wide range of endogenous and xenobiotic compounds (38) that activate AHR. Homology modeling of the AHR PAS-B domain (39) suggests that it contains a large (Ϸ500 Å 3 ) cavity used for ligand binding, supported by site-directed mutagenesis of residues in the surrounding area.…”

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

“…Mouse AhR consisting of a basic region/helix-loophelix motif, Per-Arnt-Sim domain, and Q-rich domain, possesses 17 cysteine residues, including Cys327 corresponding to human Cys333 in the ligand binding pocket of AhR (Denison et al, 2002;Li et al, 2011). Mutation of Cys327 repressed TCDD-specific binding to AhR (Pandini et al, 2007), suggesting that arylation of Cys327 may be a factor for 1,2-NQ-mediated activation of AhR. Consistent with this, mutation of this cysteine to serine diminished 1,2-NQ-mediated induction of Cyp1a1 in transfected c35 cells (Fig.…”