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    • The cavity volume of the binding pocket may

    2024-03-26

    The cavity volume of the binding pocket may differ not only between species but also among various AhR/ARNT isoforms within a single species. It is noteworthy that even a single amino Quetiapine Fumarate sale substitution within a binding pocket may result in a change in the cavity volume. This, in turn, may affect the binding affinity between TCDD and AhR-LBD. It was demonstrated in the current study that in the pig the large cavity volume of the binding pocket (804.6Å3) is accompanied by high binding affinity (−8.8kcal/mol) between TCDD and the AhR-LBD. The results of other studies seem to confirm this observation. The binding affinity between TCDD and AhR-LBD in the mouse was lower than that in the pig, and was estimated at −3.796kcal/mol with the cavity volume of 201.5Å3[3]. Similarly, in one of zebrafish AhR isoforms, the cavity volume of the binding site was established to be 600Å3 with the binding affinity of −3.972kcal/mol [3,12]. The binding pocket of the porcine AhR-LBD is strongly hydrophobic. This is caused by the presence of the following amino acids: F285, F293, P295, C298, L306, L313, G319, F322, I323, C321, M338, M346, I347, F349, L351, A365, I377 and A379. However, some polar residues were also found within the porcine AhR binding pocket, i.e., T287, H289, Y308, Y320, D327, Y334, H335, S344, S363 and Q381. The presence of hydrophilic amino acid residues alters the volume of the binding site and provides the possibility of H-bond forming, therefore affecting ligand choices. Fraccalvieri et al. [12] suggested that optimal electrostatic interactions with TCDD molecule that are needed for high affinity binding require the presence of hydrophobic as well as polar residues within the binding site of the receptor. The authors reported that the presence of polar residues (“the faced residue pair”) in the region crucial for TCDD binding in the mouse (S359-A375) and chicken (A364-S380) AhR-LBDs may stabilize the binding. In contrast, the polar residues were absent in the AhR-LBD of common tern [12] resulting in a lower binding affinity between TCDD and AhR-LBD than those in mice and chicken. In the present study, the binding affinity between TCDD and AhR-LBD was detrmined by calculating the energy of the binding (−8.8kcal/mol). Binding free energy between a ligand and protein binding pocket represents an overall energy of all the interactions within the binding pocket. This include both hydrophobic interactions and hydrogen bonds. The results of the study performed on mouse AhR [25], showed an overall binding free energy between TCDD and mAhR of −8.7kcal/mol. In the latter experiment, the binding free energy was a result of a hydrogen bonding with the energy of −8kcal/mol and hydrophobic channel calculated to have an energy of −0.5kcal/mol. In the current study a single hydrogen bond, formed between TCDD and AhR-LBD, established the ligand in the middle of the binding pocket, while a series of hydrophobic interactions stabilized the spatial orientation of the ligand. Despite the fact, that hydrogen bonds are considered to be stronger than hydrophobic interactions, the large number of amino acid residues involved in hydrophobic interactions contributes to the dominant role of these forces in stabilizing the ligand within the binding site. Moreover, it is noteworthy that TCDD is highly hydrophobic itself, and hence it interacts strongly with other hydrophobic residues. Alanine 375, demonstrated in the porcine AhR-LBD in the current study, seems to be critical for a proper ligand binding. Bisson et al. (2012) compared the TCDD sensitive mouse strain, C57BL16J with the TCDD resistant strain, DBA/2, and found that their AhR-LBDs differed at position 375. In C57BL16J mice, the 375 amino acid residue was identified as alanine, while the alanine was substituted with valine in DBA/2 mice. The authors examined in mice the effect of site-directed mutagenesis of A375 to valine or leucine on TCDD binding to AhR-LBD. The replacement of A375 with valine partially prevented TCDD binding, whereas replacement of the alanine with leucine entirely blocked the TCDD binding to AhR-LBD. Other key residues responsible for TCDD binding to the mouse AhR-LBD were also identified previously [16,30]. Mutagenesis of P291, F318 or A361 reduced or eliminated TCDD binding to AhR-LBD in mice, while mutations of C327, M334 or Q377 only partially reduced the binding [30]. In the current study, the above residues (P, F, A, C, M and Q) occurred in the constructed porcine AhR-LBD model at approximately corresponding positions.