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    • The AhR molecule is composed of multiple functional domains

    2022-11-30

    The AhR molecule is composed of multiple functional domains [38]. In the N-terminal end, the AhR contains bHLH domain which is involved in DNA binding, dimerization with ARNT and association with chaperone proteins. The PAS domain, which is situated next to the bHLH domain consists of two structural repeats: PAS-A and PAS-B [32]. PAS-A is involved in the receptor dimerization with ARNT, PAS-B, in turn, is responsible for ligand and chaperone binding [38]. Similarly to AhR, ARNT is composed of bHLH, PAS-A and PAS-B domains, and all domains are required for optimal dimerization with AhR [2]. The understanding of molecular events that underlay the ligand-AhR binding requires a detailed knowledge concerning three dimensional (3D) structures of AhR and ARNT, especially of their ligand binding domains (LBDs). In recent years, molecular modeling was successfully used to create AhR-LBD models for humans [3], mice [3,15,29], rats [3,30], rabbits [3,30], hamsters [3,30], guinea pigs [3,30], zebrafish [3], beluga whales and seals [30]. Despite the fairly broad representation of AhR-LBD models in humans or laboratory and aquatic animals, the lack of knowledge regarding farm animals is apparent. This is particularly important in view of the fact that TCDD toxicity varies among animal species. The guinea pig, for example, is much more sensitive to TCDD (LD50=0.6μg/kg of body weight), than the hamster (LD50=5000μg/kg of body weight) [36]. Such MRS 2179 tetrasodium salt sale in the animal sensitivity to TCDD may result either from differences in TCDD affinity to AhR or TCDD metabolism. The TCDD-AhR affinity, in turn, is closely associated with a structure of LBD. Therefore, the lack of information concerning TCDD-AhR-ARNT interactions in the pig justifies our interest in this species in the current study. This interest is strengthened by the significance of pigs manifested by using the species as a model animal for the study of many physiological phenomena as well as in human transplantation and nutrition. The complete mRNA sequences of the porcine AhR and ARNT which were obtained in our previous experiments ([20]; Sadowska et al., unpublished) were used to construct reliable spatial models of AhR-LBD and ARNT-LBD in the pig by means of homology modeling. To perform this analysis, the crystalline protein structures of the PAS domains of human Hif-2α [10] and ARNT [5] were used in the present study as modeling templates. The aim of the current study was to predict the structure of the porcine AhR- and ARNT-LBD by means of homology modeling. Moreover, the molecular docking was used to examine molecular interactions within the TCDD/AhR/ARNT complex. Finally, the ability of the TCDD/AhR/ARNT complex to bind to the DRE sequence was examined in porcine granulosa cells.
    Materials and methods In silico analysis (homology modeling and molecular docking) was performed on the basis of porcine AhR and ARNT cDNA sequences. The sequences were MRS 2179 tetrasodium salt sale established experimentally by next generation sequencing (NGS) and were submitted to GenBank under the following accession numbers: KM817031.1. (AhR) and KP735786.1 (ARNT). In addition, transcriptional activity of the TCDD/AhR/ARNT complex in porcine granulosa cells was examined via electrophoretic mobility shift assay (EMSA).
    Results
    Discussion The tertiary structure of the porcine AhR-LBD model generated in the current study is typical for PAS domains. This ligand binding domain was found to be formed by five anti-parallel β-sheets and several long connecting loops flanked by three α-helices. A similar model of mouse AhR-LBD was reported previously [43]. These two models revealed a high level of similarity to crystalline structure of human Hif-2α. AhR ligand binding domain seems to be well conserved among species, however sparse differences can be found. The sequence alignment of the AhR-LBD of the pig and other species demonstrated identity greater than 75%. A high sequence identity may suggest a common evolutionary path and the recent species splitting. The results published by Bisson et al. [3] showed that H285, C327, I332, M334, A375 and Q377, present in the mouse AhR-LBD, are important for proper receptor functioning and are well conserved among species. We confirmed that these residues were also present in the pig, with the exception of I332 which was found to be substituted with valine. Additionally, another mouse residue i.e., I337 was replaced with methionine in the guinea pig [3] and with valine in the pig (the current study). Finally, alanine at position 381 in the mouse was substituted with valine in the human [3] but it remains unchanged in the pig (the current study).