**2.4 AhR signaling pathways**

AhR is generally expressed in its inactive form in the cytoplasm as part of a protein complex encompassing a dimer heat shock protein, co-chaperone p23, an AhRinteracting protein, called AIP, and the protein kinase SRC (see **Figure 2**). The PAS

**Figure 2.** *Canonical pathway of aryl hydrocarbon receptor.*

*Targeting the Aryl Hydrocarbon Receptor (AhR): A Review of the In-Silico Screening… DOI: http://dx.doi.org/10.5772/intechopen.99228*

B domain within AhR binds to one monomer of the HSP90 dimer and the second HSP90 monomer interacts with the AhR basic helix–loop–helix domain (bHLH) as well as with the PAS A domain [34]. As shown in **Figure 2**, the bHLH domain within AhR is also crucial for DNA binding in a process initiated by the binding of an AhR ligand within the PAS B domain and its interaction with the co-chaperone P23. Binding to P23 stabilizes AhR in the cytoplasm, protecting it from proteasomal degrading, and also maintains the PAS B domain of AhR in a unique conformation, suitable for strong ligand binding [35, 36].

Once an AhR ligand binds to the PAS B domain, it forms an AhR-ligand complex, including p23, SRC, and AIP (see **Figure 2**). This complex is transformed into an active state and then translocated inside the nucleus. Then in the nucleus, all complex components dissociate from the AhR-ligand complex, excluding an agonist and AhR protein. Subsequently, AhR forms an active heterodimer with ARNT and creates an AhR–ARNT complex. This complex is then recruited to the DNA via the Dioxin response element (DRE), exhibiting a common DNA compromise motif (5′-TNGCGTG-3). This canonical AhR pathway increases the expression of various genes, including the principal ones in xenobiotic metabolism, AhR repressor (AHRR), and other genes [36].

#### **3. AhR three-dimensional structures**

Resolving the full-length three-dimensional structure of AhR has been a challenging exercise for the last two decades. Unfortunately, despite the many efforts towards this goal, there is no complete structure for the whole AhR protein. However, as discussed below, there are a few structures, which describe the number AhR domains. Although these structures do not reveal the exact overall AhR architecture, they can still provide useful information on the function of these separate domains. Giving computational modeling a favorable vantage point to construct reliable hypotheses for the full-length AhR organization for rational drug development and drug screening campaigns.

The first AHR 3D structure was reported in 2013 for the mouse PAS A domain (residues 110 to 267) at a resolution of 2.55 Å (PDB ID: 4M4X) (see **Figure 3**). This X-ray diffraction-based PAS A homodimer structure was obtained from recombinant *E. coli* expression. It contained a five β-sheet and elements order B-A-I-H-G, with four α-helices (Cα, Dα, Eα, and Fα) adjoining one side of the β-sheet. The interactions between the two PAS A monomers involved two distinctive areas within the A′ α-helices, revealing a strong interaction between Phe115, Leu116, and Ala119 from the A′ α-helix in one monomer with Val124, Phe260, and Ile262 from the β-sheet in the other monomer as shown in **Figure 3**. The protein structure revealed an undruggable pocket due to hydrophobic residues, and other residues such as Gln112 and Ile262 are essential in the interface for AHR dimerization, either homodimer or heterodimer with ARNT [37, 38].

Two more additional AhR structures were revealed in 2017 (see **Figure 4**). The two structures comprise multiple AhR domains and show a clear interaction between AhR and its dimerization partner, ARNT, as well as its interaction with two DNA strands. The two structures (PDB IDs: 5V0L and 5NJ8) [39, 40] were resolved at a resolution of 4.0 and 3.35 Å, respectively and revealed the complex formation among the bHLH and PAS A domains from human AhR and their interactions with ARNT and DNA. However, due to the observed high flexibility of the AhR PAS B domain and the transactive domain (C- terminal), none of these two subdomains were included in this architecture. However, both structures clearly explain the protein–protein interactions (PPI) and show clear interface regions for these

#### **Figure 3.**

*The upper figure represents crystal structure of homodimer mouse AhR-PAS a obtained from protein databank [37], and the lower figure shows the interactions between the two monomers.*

interactions between the individual domains within AhR as well as their interactions with ARNET and DNA.

As shown in **Figure 4**, the first PPI interface is between the AhR-ARNT heterodimer with the two DNA strands. This interaction is mediated by DRE Ser36, His39, and Arg40 from the AhR bHLH domain and His79, Asp83, Arg86, and Arg87 from ARNT, as well as thymine and guanine from the DNA. The second PPI interface is between AhR and ARNT through different regions within the two proteins. These regions involve many hydrophobic interactions from both proteins and comprise residues Leu47, Leu50, Leu53, Val74, and Leu70 from the AhR bHLH domain and residues Ile109, Leu112, Val136, and Met139 in ARNT. The third PPI interface involves interactions between residues from the PAS A domain in both AhR and ARNT, mediated by residues Phe117, Leu118, Ala121, Leu122, Tyr137, Val126, Phe266, and Ile268 from AhR. The fourth, and final PPI interface encompasses the interdomain interactions between the AhR bHLH

*Targeting the Aryl Hydrocarbon Receptor (AhR): A Review of the In-Silico Screening… DOI: http://dx.doi.org/10.5772/intechopen.99228*

**Figure 4.**

*Crystal structure of human aryl hydrocarbon receptor in heterodimer with aryl hydrocarbon nuclear translocator and recruit on DNA in dioxin element response [39].*

and AhR PAS A domains, through residues Phe136, Ser151, Ile154, and Leu246 from the PAS A domain and Phe56, Val60, Leu72, Ala79, and Phe82 from the bHLH domain.
