A model for recognition of polychlorinated dibenzo- p -dioxins by the aryl hydrocarbon receptor M. Procopio 1 , A. Lahm 2 , A. Tramontano 3 , L. Bonati 1 and D. Pitea 1 1 Dipartimento di Scienze dellÕAmbiente e del Territorio, Universita Á degli Studi di Milano-Bicocca, Milano, Italy; 2 Istituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia (Roma), Italy; 3 Dipartimento di Scienze Biochimiche ÔRossi FanelliÕ, Universita'di Roma ÔLa SapienzaÕ, Roma, Italy Ligand binding by the aryl hydrocarbon receptor (AhR), a member of the bHLH-PAS family of transcriptional reg- ulatory proteins, has been mapped to a region within the second ÔPASÕ domain, a conserved sequence motif ®rst discovered in the P er-ARNT-Sim family of pr oteins. In addition to the bacterial photoactive yellow protein (PYP), which had been proposed as a structural prototype for the three dimensional fold of PAS domains, two crystal structures of the P AS domain have recently been deter- mined: the human potassium channel HERG and the heme binding domain of the bacterial O 2 sensing FixL protein. The three structures reveal a highly conserved structural framework in e volutionary rather distant PAS domains, p rovide a more g eneral view of how these domains can recognize their ligands and suggest a structure±function relationship that we exploited to build a three-dimensional model of the ligand binding domain (LBD) of the mouse aryl hydrocarbon receptor (mAhR). The model allowed us to putatively i dentify the residues responsible for the recognition of polychlorinated dibenzo- p-dioxins (PCDDs) by AhR receptors and to formulate an hypothesis on the signal transduction mechanism. Keywords: aryl h ydrocarbon receptor; polychlorinated dibenzo-p-dioxins; structure prediction; protein modelling; molecular recognition. Studies on the biological mechanism of action of polychlo- rinated dibenzo-p-dioxins (PCDDs) indicate that their biological effects are mediated by binding to a speci®c cytoplasmic protein, the aryl hydrocarbon receptor (AhR) [1]. Ligand-induced activation of AhR initiates a process whereby t he receptor i s transformed into a nuclear transcription factor by forming a complex with the protein ARNT (Ah receptor nuclear translocator). Speci®c recog- nition of XRE DNA sequences (xenobiotic responsive elements) by the ligand-activated AhR/ARNT heterodimer then induces transcription of genes encoding xenobiotic metabolizing enzymes [2]. Understanding the PCDD±AhR binding process at a molecular level is therefore a key step for gaining insight into the biological mechanism of action of these compounds. The structure±activity relationship (SAR) of the PCDD± AhR interaction has been studied with th e aim of correlat- ing physico-chemical properties o f the ligand s and their biological activities [3±5]. In particular, we analysed a series of PCDDs with varying binding af®nities [4,5] on t he basis of their molecular electrostatic potential (MEP) and molec- ular polarizability and concluded that the requirements f or high af®nities are the concentration o f negative M EP values at the e xtremes of t he ligand's long axis and a d epleted charge above and below the aromatic rings. This led to the hypothesis that there are favorable interactions with a receptor nucleophilic site in the central part of the ligand and with electrophilic sites at both sides of the principal molecular axis. A necessary next step to understand the PCDD±AhR interaction and t o identify the amino-acid residues directly inte racting w ith P CDDs is the construction of a three-dimensional model for the AhR ligand binding domain (LBD). AhR and ARNT belong to the Per-ARNT-Sim (PAS) family of proteins [6,7], whose members act as transcrip- tional activators, sensor modules of two-component regu- latory sy stems o r a s ion channels [8]. PAS domains are found predominantly in p roteins that are involved, directly or indirectly, in signal transduction. Their known functions are in some cases to mediate protein±protein interactions and, in other cases, s uch as f or AhR, ligand a nd/or cofactor binding [8]. In AhR, two PAS domains are present in a 270-residue region encompassing two imperfect repeats of  110 amino acids ( PAS-A and PAS-B) separated by a sequence of  50 amino acids. A minimal LBD was mapped in the mouse AhR (mAhR) between amino acids 230 and 397, the region that encompasses the PAS-B repeat [9]. While deletion of the PAS-A repeat (amino acids 121±182) reduced ligand binding only to 30%, deletion of the PAS-B repeat (amino acids 259± 374) completely abolished binding, as did deletion Correspondence to A.Tramontano,DepartmentofBiochemical Sciences ÔRossi FanelliÕ, University of Rome ÔLa SapienzaP.le Aldo Moro, 500185 Rome. Fax: + 39 06 91093482, Tel.: + 39 06 91093207, E-m ail: Anna.Tramontano@uniroma1.it Abbreviations: AhR, aryl hydrocarbon receptor; PYP, photoactive yellow protein; LBD, ligand binding domai n; mAhR, mouse aryl hydrocarbon receptor; PCDD, polychlorinated dibenzo-p-dioxin; ARNT, Ah rec e ptor nu clear t r anslocator; XRE, xenobiotic responsive elements; SAR, structure±activity relationship; MEP, molecular electrostaticpotential;PAS,Per-ARNT-Simproteinfamily;TCDD, tetrachlorodibenzo-p-dioxin. (Received 10 August 2001, revised 20 September 2001, accepted 16 October 2001) Eur. J. Biochem. 269, 13±18 (2002) Ó FEBS 2002 of the complete PAS region [9]. In the same study it was already shown that modi®cations outside the PAS domain had no effect on ligand binding. A s tructural prototype for the three-dimensional fold of the PAS domain superfamily has been proposed to be the structure of the photoactive yellow protein (PYP), a bacterial light-sensing protein [10]. However, the crystal structures of two other PAS domains [11,12] have been recently determined and their analysis a llowed us t o build a three-dimensional model of the mAhR LBD and to investigate its ligand binding site at the molecular level. RESULTS AND DISCUSSION Structure prediction Application of a re cursive PSI - BLAST [13] search (default parameters) against the nonredundant protein sequence database revealed a high number of matches between the mAhR LBD and many other PAS proteins, including hypoxia-inducible factor 1a, several histidine kinases, light receptors, regulatory proteins, clock proteins (such as the period clock protein PER), sensor proteins (oxygen/redox sensors) and ion channels. T he crystal structures of t he PAS domains of two of these proteins were recently solved: the human potassium channel HERG [11] and the heme binding domain of the bacterial O 2 sensing FixL protein [12]. Both structures were detected after four (HERG) or eight (FixL) PSI - BLAST iteration cycles, as was the PYP protein (iteration 6). Although E values w ere initially rather high (> 0.1) for all three structures, E values for HERG and FixL became highly signi®cant (< 10 )4 )asthesearch progressed. A search including only the database of known protein structures neither found any of these structures, nor highlighted any other statistically signi®cant homologies. The structures of the HERG and FixL P AS domains are showninFig.1togetherwiththePYPstructure.Giventhe low level of sequence identity [15], the high structural conservation is quite unexpected: all the structures are formed by a ®ve-stranded antiparallel b sheet with a helices on one side. Although all three domains belong to proteins involved in signalling p rocesses a nd are expected t o transmit a signal through protein±protein i nteractions, they have developed quite different mechanisms to perform their function. While the HERG PAS domain does not bind a ligand [11], both the FixL PAS domain and PYP are activated by ligands; in FixL, oxygen binding at the heme binding PAS domain controls the activity of a h istidine kinase domain [12]; and in PYP, a local conformational change occurs once the p-hydroxycinnamoyl cromophore is bound [10]. The largest conformational difference between the FixL and the HERG and PYP structures occurs in the so-called helical connector, the long central helix, which sh ows a translational displacement of  7A Ê that allows the accom- modation of the heme cofactor (Fig. 1C) [12]. The hydro- phobic core of the three domains is generally well conserved, but two buried residues in FixL differ signi®cantly in size from the structurally equivalent residues in PYP and HERG, again favoring the heme binding. These are g lycines 224 and 251 that substitute Phe96 a nd Val120 in PYP, and Phe98 and Leu127 in HERG [16]. For both FixL and PYP, structures are known for the inactive and active signaling states. In the case of PYP, conformational changes occur in the neighborhood of the p-hydroxycinnamoyl cromophore and are transmitted to the s urface of the protein primarily through t he cromophore and Arg52 [10]. In FixL, the heme propionate groups are suggested to relay the spin transition signal by transducing the increased planarity of the pu ckered porphyrin ring into backbone and side-chain conformational changes within a loop (residues 211±215) immediately following the helical connector [12]. The suggested signal transducing regions of PYP and FixL are thus located at the opposite ends of the Fig. 1. Schematic representation of the HERG (A), PYP (B), and FixL (C) PAS domains displaying the high degree of structural similarity. The largest shift amongst the conserved s econdary element position o ccurs i n FixL due t o the presence of the large heme c ofactor. Secondary structure elements are colored blue (strands ) and red (helices), cofactor ligands gr een. (A), (B) and (C) were generat ed using RIBBONS [28]. Coordinate sets used correspond to entries 1BV5(FixL) [12], 2PYP(PYP) [ 10] and 1BYW(HERG) [11] of the PDB protein data bank [14]. 14 M. Procopio et al. (Eur. J. Biochem. 269) Ó FEBS 2002 long central he lix, h ighlighting th e imp ortance of this region and t he ¯anking loops as the critical regulatory region of the PAS domain family [16], with the remainder of the PAS fold serving as a structural scaffold. When sequence s imilarity between the t arget a nd potential template sequences is low, as in our case, t he correctness of the alignment plays a crucial role both in the selection of the correct template and in the expected ®nal quality of the model. Other information, such as predicted and observed secondary structures of the target and template proteins, and sequence and structure conservation in their families, should therefore be used to re®ne the alignment. The consensus secondary structure for residues 230±397 of th e mAhR LBD a s predicted by the JPRED server [17,18] is reported i n Fig. 2 together with the o bserved s econdary structure of the three template candidates. The ®nal sequence alignment used for modelling is reported in the same ®gure. This sequence alignment differs somehow from PAS domain alignment recently p roposed [8], as it was produced manually taking into account th e predicted secondary structure of AhR LBD, the observed secondary structure and FSSP structural alignment for FixL, HERG and PYP, and the c onservation pattern in a m ultiple alignment of AhR sequences. For clarity, in Fig. 2 we only show some of these latter sequences and, for comparison, a subset of sequences from the related ARNT proteins. The AhR sequences in Fig. 2 w ere selected for their differences in the response to PCDDs: the human Ah receptor that has an af®nity fo r 2,3,7,8-TCDD sixfold lower than mAhR [19]; the AhR-1 ortholog of Caenorhabditis elegans (AhR-1C.E.), neither photoaf®nity labeled by a dioxin analog, nor activated by b-naphto¯avone in a yeast system [20]; the rainbow trout AhRa that binds TCDD [21] and t he Microgadus Tomcod AhR also activated by TCDD [22]. All alignments were manipulated using the interactive display program SEAVIEW [23]. Fig. 2. Alignment of Ah receptors and their predict ed secondary structure against the three structural templates a ligned a ccording to FSSP. a Helices and b strands are represented as white and black b ars, respectively. Secondary structure assignment for FixL, PYP a nd HERG is derived from the PDB entries. Colouring sc heme for resid ues: red: acidic ; blue: basic; purple : polar; yellow: Cys; brown : aromatic; gree n: hydrophob ic; orange: Ser,Thr; gr ey: Pro; wh ite: Gly. Ó FEBS 2002 A model for PCDD ± AhR recognition (Eur. J. Biochem. 269)15 Modelling Because of the closer functional homology (noncovalent interaction with a ligand) we used FixL as a template for modelling. This choice was also motivated by the observa- tion that the helical connector in FixL is translated away from the b sheet with respect to HERG and PYP (Fig. 1) thus allowing binding of the heme-ligand, a situation expected to be present in a similar fashion also in AhR. The sequence corresponding to mAhR residues 275±380 was therefore inscribed onto the structural template pro- vided by FixL a ccording to the alignmen t shown in F ig. 2 , and, subsequently, the necessary i nsertions and d eletions were modeled (Fig. 3A). AhR residues 381±397 were not modeled b ecause the corresponding helix in FixL is pointing away from the barrel and should not be involved in ligand binding. AhR residues 286±288 c ould be modeled using the corresponding loop of equal length from the HERG structure, while all other insertions and deletions were constructed u sing a fragment database s earch procedure [24]. The one-residue insertion at position 314 c orrelates well with the presence of a hydrophilic residue at the spatially close position 282 replacing the buried hydrophobic Ile present in FixL. Fig. 3. Comparison between the mAhR model (A,C) and the parental FixL PAS d omain structure (B,D). In (A) a nd (B) r esidu es tha t in¯ue nce the size of the ligand p ocket a re h ighligh ted. The arrow indicates t he shift of the he lical co nn ector ( orange) i n Ah R with resp ect t o F ixL. Lo op region s w here insertions or deletions had to be accomodated in the AhR model are coloured magenta. In (C) and (D) a close-up of the AhR and FixL ligand binding po ckets is sh own. T he k ey e lements in the proposed signal t ransduct ion m echanism fo r FixL, a change in side-chain conformation of Arg206, Thr210 and Asp212, are conserved in AhR with Arg333, Thr337 and Glu339 equivalently positioned, r eady to sense and transduce the presence of the PCDD ligand . Addition al AhR residues involved i n ligand reco gnition and discrimin ation a re Arg282 and Gln377 close to t he polar end of the ligand inside the pocket. TCDD and heme cofactor atoms are colored green (carbon), red (oxygen), blue (nitrogen), yellow ( iron) and magenta (chlorine). 16 M. Procopio et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The postulated signaling loop of FixL (amino acids 211± 215) following the helical connector had to be substituted by a s horther frag ment in mAhR (Fig . 2, a mino acids 335±338) that could only be achieved in our model by manually shifting the helix towards the b sheet. A l ast insertion occurred at G ly368, in a loop region unlikely to be involved in interactions with t he ligand. The backbone geometry of the resulting model was regularized with WHAT IF [25], the side-chain rotamers of substituted residues optim ized using SCWRL [26] and the model analyzed without any further modi®cations. A model for recognition of PCDDs by Ah receptors The most noticeable conformational difference between the mAhR model a nd the FixL template is the relative position of the helical connector that moves closer to the b sheet, thus reducing t he size of the binding cavity entrance (Fig. 3). The helix position, intermediate between that observed in HERG and in FixL, correlates well with the functional role of the hydrophobic core in the three proteins, while HERG lacks any binding activity; the modeled mAhR binds PCDDs and FixL has to accomodate the larger heme cofactor. The AhR residues at positions important for heme binding in FixL support our model. Gly224 and Gly251 in the hydrophobic core of FixL correspond to Leu347 and Ala375 in mAhR thus reducing the size of the cavity. This is also consistent with site-directed mutagenesis r esults that identi®ed Ala375 as critical for the ligand binding activity [19]. Interestingly, there is also a good correlation between the size of the side-chain at this position and the size of the ligand. While the latter decreases from FixL to AhR to HERG, the side-chain volume increases (from Gly251 to Ala375 to Leu127). Moreover, human AhR and AhR-1C.E., both with reduced af®nity for PCDDs, have bigger side-chains at this position (Val and Leu, respectively) partially ®lling t he binding cavity. The residue coordinating the f erric heme ion in FixL, His200, is substituted by Cys327 in all AhR receptors, except for AhR-1C.E. where methi- onine is present. At the entrance of the FixL ligand cavity, Arg220, that binds a heme propionate group, is replaced by Thr in all AhR (Thr343 in mAhR), except for human AhR and AhR-1C.E. that have isoleucine and leucine, respectively. While the CG2 methyl group of Thr could m ediate hydro- phobic interactions with the ligand, both i soleucine and leucine will partially block the entrance and reduce af®nity. None of these residues, characteristic of the AhR proteins, are conserved in the homologous ARNT proteins (Fig. 2) which have no ligan d binding activity. Additional information ab out the PCDD±AhR binding can be deduced by analyzing the proposed mechanisms for signal transduction of FixL [12,16,27]. A ccording to Perutz et al. [27], the pathway s tarts at Ile215, Leu236, Ile238, which form a hydrophobic triad around the heme ligand. The movements of these residues are transmitted to, and ampli®ed by, a loop that includes Pro213, and then transmitted to other atoms including the h eme p ropio- nates. A different key residue has been proposed by Gong et al. [12] who indicated the interaction between heme propionates and His (or Arg) 214 as the starting event of the protein conformational change. It has also been observed that, going from the unbound to the bound state, Arg206 affects the position of Asp212, which in turn undergoes the largest c onformational change o f all the sidechains [12]. Interestingly, although the conformation of the signaling loop had to be altered in the mAhR model, Arg206 and Thr210 of FixL are in e quivalent structural positions as Arg333 and Thr337 in mAhR and Asp212 of FixL is replaced by the very s imilar Glu339. These three r esidues are conserved in all Ah receptors and are not present in the other PAS proteins analyzed. Therefore, by analogy with the FixL mechanism, it is conceivable that, once PCDD is bound, Arg333 in mAhR is involved in the interaction with one of the chlorine atoms and breaks the hydrogen bond with Glu339 that changes conformation. The ligand with t he highest af®nity for the AhR is 2,3,7,8- TCDD and our model can be used to investigate its mode of binding, under t he assumption that the molecular plane of TCDD is in a similar position as that o f the heme group in FixL. We highlight in Figs 3C,D, the residues predicted to mediate k ey ligand interactions in the proposed binding cavity. The size of Ala375 is important for ligand accom- modation, Cys327 co uld interact with the electrophilic central region of TCDD [4], Thr343 possibly stabilizes the complex by hydrophobic interactions, Arg333, at the entrance of the cavity, may guide TCDD t owards its binding site by long-range electrostatic interactions and, by interacting with chlorine atoms of TCDD, may promote a signal transduction mechanism through Glu339, similar to that of FixL. Two additional residues, Arg282 and Phe345, are shown in the Fig. 3. While Arg282, replaced by Gln in some Ah receptors and pointing t o the TCDD chlorinated side, may contribute to the binding by electrostatic interac- tions or hydrogen bond, Phe345, lining one side of the ligand pocket, could interact with the aromatic ringsystem of TCDD. Ultimately, Gln377, characteristic of all Ah receptors and not present i n o ther PAS proteins, could f orm hydrogen bonds w ith chlorine atoms in the predicted binding cavity for TCDD. Most of the proposed interactions ®t well with the electrostatic characteristics we highlighted in previous QSAR studies on ligand properties [4,5]. The requirements of a nucleophilic site in the central part of the ligand and of electrophilic sites at the sides of the principal molecular axis are both explained by our model. CONCLUSIONS Given the limitations in today's modelling and prediction techniques, the model presented here has to be considered only an a pproximate and probably i ncomplete picture of the ligand binding domain of AhR and of its interactions with PCDDs. It should also be emphasized that the LBD is part of a much larger protein and some features of the Ah receptor system might not be explainable in terms of the isolated domain. PYP has been previously proposed as an app ropriate structural template for AhR, but our analysis of the recently determined structures of the FixL and the HERG P AS domains strongly suggests that a model based on FixL is more likely to be correct. On one hand, the availability of the t hree structures indicates that the position of the helical connector can differ. On the other, the closer functional homology between FixL and AhR, the secondary structure Ó FEBS 2002 A model for PCDD ± AhR recognition (Eur. J. Biochem. 269)17 prediction and the size of the ligand all point to FixL as a more suitable candidate. Our model, although based on low sequence similarity, is capable of explaining all known experimental and theoret- ical data and therefore we believe it to be accurate enough t o serve as a framework for furt her experiment s such as site directed mutagenesis of residues proposed to mediate the AhR±PCDD interaction and docking calculations to more accurately de®ne the orientation of the ligand i n the binding cavity. ACKNOWLEDGEMENTS The ®nancial support by the Italian CNR (grant no. 98.03245.ST74) and the Fondazione Lomb ardia per l'Ambiente is gratefully acknowl- edged. REFERENCES 1. Landers, J.P. & Bunce, N.J. (1991) The Ah receptor and the mechanism of dioxin t oxicity. Biochem. J. 276, 273±287. 2. Sogawa, K. & Fujii-Kuriyama, Y. (1997) Ah receptor, a novel ligand-activated transcription factor. J. Biochem. 122, 1075±1079. 3. Waller, C.L. & Mckinney, J.D. (1995) Three-dimensional quan- titative structure-activity relationships of dioxins and dioxin-like compounds: model validation and Ah receptor characterization. Chem. Res. Toxicol. 8, 847±858. 4. Bonati, L., Fraschini, E., Lasagni, M., Modoni, E.P. & Pitea, D. (1995) A h ypothesis o n t he me chanism o f P CDD b iological activity based on molecular electro static potential modeling. Part 2. J. Mol. Struct. (THEOCHEM) 340, 83±95. 5. Fraschini, E., Bonati, L. & Pitea, D. (1996) Molecular polariz- ability as a tool for u nderstan ding the b in ding properties of polychlorinated dibenzo-p-dioxins: de®nition of a reliable com- putational procedure. J. Phys. C hem. 100, 10564±10569. 6. Hahn, M.E., (1991) The a ryl hydrocarbon re ceptor: a comparative perspective. Comp. Biochem. Physiol. Part C 121, 23±53. 7. Gu, Y.Z., Hogenesch, J.B. & Brad®eld, C.A. (2000) The PAS superfamily: sensors of environmental and developmental signals. Annu. Rev. Pharmacol. Toxicol. 40, 519±561. 8. Taylor, B.L. & Zhulin, I.B. (1999) PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol. Mol. Biol. Rev. 63, 479±506. 9. Fuk unaga, B.N., Probst, M.R., Reisz-Porszasz, S. & Hankinson, O. (1995) Identi®cation of functional domains of the aryl hydro- carbon receptor. J. Biol. Chem. 27 0, 29270±29278. 10. Pellequer, J.L., Wager-Smith, K.A., Kay, S.A. & Getzo, E.D. (1998) Photoactive yellow protein: a structural prototype for the three-dimensional fold o f the PAS domain superfamily. Proc. Natl Acad. Sci. USA 95, 5884±5890. 11. Cabral, J.H.M., Lee, A., Cohen, S.L., Chait, B.T., Li, M. & Mackinnon, R. ( 1998) Crystal structure an d functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain. Cell 95, 649±655. 12. Gong, W., Hao, B., Mansy, S.S., Gonzalez, G., Gilles-Gonzalez, M.A. & Chan, M.K. (1998) Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction. Proc. Natl A cad. Sci. USA 95, 15177±15182. 13. Altsc hul, S .F., Madden, T .L., S cha È er, A.A., Z hang, J ., Zhang, Z., Miller, W. & Lipman, D.J. (1997) Gapped BLAST and PSI- BLAST: a ne w generation o f p rotein database search programs. Nucleic Acids Res. 25, 3 389±3402. 14. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., B hat, T.N., Weissig, H., Shindyalov, I.N. & Bourne, P.E. (2000) The p rotein data bank. Nucleic Acids Res. 28, 235±242. 15. Holm, L. & Sander, C. (1996) Mapping the protein universe. Science 273, 595±602. 16. Pellequer, J.L., Brudler, R. & Getzo, E.D. (1999) Biological sensors: more than one way to sense oxygen. Current Biol. 9, R416±R418. 17. Cu, J .A., Clamp, M.E., Siddiqui, A.S., Finlay, M. & Barton, G .J. (1998) JPred: a consensus secondary structure prediction server. Bioinformatics 14 , 892±893. 18. Cu, J.A. & Barton, G.J. (1999) Evaluation and improvement of multiple sequence methods for protein secondary s tructure prediction. Proteins 34, 508±519. 19. Ema,M.,Ohe,N.,Suzuki,M.,Mimura,J.,Sogawa,K.,Ikawa,S. & Fujii-Kuriyama, Y. (1994) Dioxin binding activities of poly- morphic forms of mouse and human arylhydrocarbon receptors. J. Biol. C hem. 269, 27337±27343. 20. Powe ll-Coman, J.A., Brad®eld, C.A. & W oo d, W.B. (1998) Caenorhabditis elegans orthologs of the aryl hydrocarbon recep- tor and its heterodimerization partner the aryl hydrocarbon receptor nu clear translocator. Proc. Natl Acad. Sci. USA 95, 2844±2849. 21. Abnet, C.C., Tanguay, R.L., Hahn, M.E. & W.Peterson, R.E. (1999) Two forms of aryl hydrocarbon receptor type 2 in rainbow trout (Oncorhync hus mykiss). Evidence for dierential expression and enhancer speci®city. J. Biol. Chem. 274, 15159±15166. 22. Roy, N.K. & Wirgin, I. (1997) Characterization of the aromatic hydrocarbon r ecepto r gene and i ts expression in Atlantic tomcod. Arch. Biochem. Biophys. 344, 373±386. 23. Galtier, N., Gouy, M. & Gautier, C . (1996) SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosc. 12, 543±548. 24. Molecular Simulation Inc. (1998) INSIGHT II Molecular Simu- lation Inc. San D iego, CA, USA. 25. Vriend, G. (1990) WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52±56. 26. Bower, M.J., Cohen, F.E. & Dunbrack, R.L. Jr (1997) Prediction of protein side-chain rotamers from a backbone-dependent rot- amer library: a new homo logy modeling t ool. J. Mol. Biol. 267, 1268±1282. 27. Perutz, M.F., Paoli, M. & Lesk, A.M. (1999) Fix L, a haemo- globin that acts as an oxygen sensor: signalling mechanism and structural basis of its homology with PAS domains. Chem. Biol. 6, R291±R297. 28. Carson, M. (1991) RIBBONS 2.0. J. Appl. Cryst. 24, 958±961. 18 M. Procopio et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Roma, Italy Ligand binding by the aryl hydrocarbon receptor (AhR), a member of the bHLH-PAS family of transcriptional reg- ulatory proteins, has been mapped. ortholog of Caenorhabditis elegans (AhR-1C.E.), neither photoaf®nity labeled by a dioxin analog, nor activated by b-naphto¯avone in a yeast system [20]; the rainbow