The Ah receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and naturally-occurring chemicals. Although a significant amount of information is available with respect to the planar aromatic hydrocarbon AhR ligands, the actual spectrum of chemicals that can bind to and activate the AhR is only now being elucidated. In addition, the lack of information regarding the actual three-dimensional structure of the AhR ligand binding domain (LBD) has hindered detailed analysis of the molecular mechanisms by which these ligands bind to and active AhR signal transduction. In this review we describe the current state of knowledge with respect to naturally occurring AhR ligands and present and discuss the first theoretical model of the AhR LBD based on crystal structures of homologous PAS family members.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent, basic helix-loop-helix Per-Arnt-Sim (PAS)-containing transcription factor that can bind and be activated by structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Our previous three-dimensional homology model of the mouse AhR (mAhR) PAS B ligand binding domain allowed identification of the binding site and its experimental validation. We have extended this analysis by conducting comparative structural modeling studies of the ligand binding domains of six additional high-affinity mammalian AhRs. These results, coupled with site-directed mutagenesis and AhR functional analysis, have allowed detection of the "TCDD binding-fingerprint" of conserved residues within the ligand binding cavity necessary for highaffinity TCDD binding and TCDD-dependent AhR transformation DNA binding. The essential role of selected residues was further evaluated using molecular docking simulations of TCDD with both wild-type and mutant mAhRs. Taken together, our results dramatically improve our understanding of the molecular determinants of TCDD binding and provide a basis for future studies directed toward rationalizing the observed species differences in AhR sensitivity to TCDD and understanding the mechanistic basis for the dramatic diversity in AhR ligand structure.The aryl hydrocarbon receptor (AhR) 1 is a ligand-dependent transcription factor and a member of the basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family of proteins (1-4). † This research was supported by the National Institutes of Environmental Health Sciences (Grants ES07685 and ES012498) and the California Agricultural Experiment Station.*To whom correspondence should be addressed. Phone: (530) 752-3879. Fax: (530) 752-3394. msdenison@ucdavis.edu. SUPPORTING INFORMATION AVAILABLE Expression levels of in vitro synthesized wild-type and mutant AhRs. 35 S-Labeled wild-type (wt) and mutant AhRs (specific mutation indicated) were synthesized in vitro, denatured, and resolved by SDS-polyacrylamide gel electrophoresis and phosphoimager analysis of the dried gels as described in Materials and Methods (Figure 1). This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations: AhR, aryl hydrocarbon receptor; bHLH, basic helix-loop-helix; PAS, Per-Arnt-Sim; LBD, ligand binding domain; HAH, halogenated aromatic hydrocarbon; PAH, polycyclic aromatic hydrocarbon; TCDD,2,3,7, hsp90, heat shock protein of 90 kDa; XAP2, X-associated protein 2; ARNT, AhR nuclear translocator; HIF-2α, hypoxia-inducible factor 2α; DRE, dioxin responsive element; mAhR, mouse AhR; rtAhR, rat AhR; haAhR, hamster AhR; rbAhR, rabbit AhR; gpAhR, guinea pig AhR; bAhR, beluga whale AhR; sAhR, seal AhR. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 30. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWhile the highest-affinity AhR ligands include persistent environmental co...

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 ...

Allostery offers a highly specific way to modulate protein function. Therefore, understanding this mechanism is of increasing interest for protein science and drug discovery. However, allosteric signal transmission is difficult to detect experimentally and to model because it is often mediated by local structural changes propagating along multiple pathways. To address this, we developed a method to identify communication pathways by an information-theoretical analysis of molecular dynamics simulations. Signal propagation was described as information exchange through a network of correlated local motions, modeled as transitions between canonical states of protein fragments. The method was used to describe allostery in two-component regulatory systems. In particular, the transmission from the allosteric site to the signaling surface of the receiver domain NtrC was shown to be mediated by a layer of hub residues. The location of hubs preferentially connected to the allosteric site was found in close agreement with key residues experimentally identified as involved in the signal transmission. The comparison with the networks of the homologues CheY and FixJ highlighted similarities in their dynamics. In particular, we showed that a preorganized network of fragment connections between the allosteric and functional sites exists already in the inactive state of all three proteins.—Pandini, A., Fornili, A., Fraternali, F., Kleinjung, J. Detection of allosteric signal transmission by information-theoretic analysis of protein dynamics.

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-protein docking is increasingly used in Drug Discovery. The initial limitations imposed by a reduced availability of target protein structures have been overcome by the use of theoretical models, especially those derived by homology modeling techniques. While this greatly extended the use of docking simulations, it also introduced the need for general and robust criteria to estimate the reliability of docking results given the model quality. To this end, a large-scale experiment was performed on a diverse set including experimental structures and homology models for a group of representative ligand-protein complexes. A wide spectrum of model quality was sampled using templates at different evolutionary distances and different strategies for targettemplate alignment and modeling. The obtained models were scored by a selection of the most used model quality indices. The binding geometries were generated using AutoDock, one of the most common docking programs. An important result of this study is that indeed quantitative and robust correlations exist between the accuracy of docking results and the model quality, especially in the binding site. Moreover, state-of-the-art indices for model quality assessment are already an effective tool for an a priori prediction of the accuracy of docking experiments in the context of groups of proteins with conserved structural characteristics.

BackgroundThe hierarchical and partially redundant nature of protein structures justifies the definition of frequently occurring conformations of short fragments as 'states'. Collections of selected representatives for these states define Structural Alphabets, describing the most typical local conformations within protein structures. These alphabets form a bridge between the string-oriented methods of sequence analysis and the coordinate-oriented methods of protein structure analysis.ResultsA Structural Alphabet has been derived by clustering all four-residue fragments of a high-resolution subset of the protein data bank and extracting the high-density states as representative conformational states. Each fragment is uniquely defined by a set of three independent angles corresponding to its degrees of freedom, capturing in simple and intuitive terms the properties of the conformational space. The fragments of the Structural Alphabet are equivalent to the conformational attractors and therefore yield a most informative encoding of proteins. Proteins can be reconstructed within the experimental uncertainty in structure determination and ensembles of structures can be encoded with accuracy and robustness.ConclusionsThe density-based Structural Alphabet provides a novel tool to describe local conformations and it is specifically suitable for application in studies of protein dynamics.

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