## 3 Results and Discussion
### Docking Analysis
#### 3.2.3 Molecular Interactions of CYP3A4 with AFB1 Using Different PDB IDs  
Because of its high toxicity and carcinogenic ability, AFB1 is the most studied/reported aflatoxin. Due to the double bond in the AFB1 structure, this molecule presents an epoxidation reaction within the cytochrome. The resulting epoxidized aflatoxin can form adducts with DNA. On the other hand, several authors describe different amino acids and conformations in the active site of the cytochrome [28; 30; 31]. For these reasons, only AFB1 was used in the simulations of the molecular interactions with CYP3A4 using three different files: 2V0M, 4 NY4, and 1W0E PDBs. This exploration was made in order to account for these differences within the active site. The calculations were carried out using the M06-2X/6-311G(d,p) level of theory because the first docking analysis of the aflat B1, B2, G1, and G2 with the proteins CYP3A4 (1 TQN) and CYP1A2 (2 HI4) showed the most stable binding energy when this functional was applied. In our computed models, remarkable differences were found in the conformation of the active site and the orientation of the ligand. Table 4 and Figure 5 show the results, using the amino acids of the active site of 1 TQN docking as reference. Figure 4: Analysis of the molecular interactions in the active site of CYP1A2 (2 HI4 PDB ID) with ligands AFB1, AFB2, AFG1, and AFG2. Hydrogen bonds are indicated with green dotted lines. [PERSON] et al., in 2017 [28], developed a docking analysis of the interaction between AFB1 and CYP3A4, finding different conformations depending on the PDB ID file used in the calculations. The same behavior was observed in our results. The binding pocket could change depending on the ligands included in the original crystallographic structure of the selected protein [8; 32]. In another study, using 2V0M PDB ID, it was reported that the interaction with aflatoxin B1 in the active site occurred between the amino acid residues of Leu210, Leu211, and Phe304. These residues are indispensable for the positive homotropic cooperativity of oxidation [30]. However, in our results, these amino acids were not present in the active site. [PERSON] et al., in 2013 [31], reported Phe108, Ser119, Ile120, Leu211, Asp214, Ile301, Phe304, Ala305, Thr309, Ala370, and Leu373 as residues involved in the substrate binding, cooperativity, and regioselectivity of CYP3A4. Likewise, [PERSON] reported that the residues Ser119, Arg212, and Thr224 provide polar contacts. As we can see in Table 4, this information is consistent with the amino acids found in the active sites of the different PDBs studied in this work. In a previous study, using the CYP3A29 of pig (a human CYP3A4 homolog), it was reported that Thr309 is a residue essential to the correct orientation of AFB1 epoxidation through hydrogen bonding [33]. Our results concur on the role of this amino acid; however, a hydrogen bond was not observed in the interaction. The stability of the conformation for 1 TQN PDB was given by \(\pi\)-\(\pi\) interactions between the central aromatic ring of AFB1 and the five rings of the HEM group (see Figure 5A). For 1W0E PDB, no hydrogen bonds or \(\pi\)-\(\pi\) interactions were detected (see Figure 5C). In the case of 4 NY4 PDB, a hydrogen bond (Leu 483-O3) was observed (see Figure 5D), whereas for 2V0M \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **1 TQN** & **2V0M** & **1W0E** & **4 NY4** \\ \hline \multirow{4}{*}{Active Site} & Arg105, Ser119, & \multirow{4}{*}{Arg105, Ser119, } & \multirow{4}{*}{Ser119, Arg212, } & \multirow{4}{*}{Arg212, Thr309, } \\  & Arg212, Ala305, & & \\ Thr309, Ala370, & & & \\ Arg372, Leu373, & & & \\ Glu374, HEM508 & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Analysis of the active site in CYP3A4 (using different PDB IDs) after interaction with aflatoxin AFB1, provided by molecular docking simulations. Figure 5: Analysis of the molecular interactions between aflatoxin AFB1 and CYP3A4 (using different PDB ID files): (**A**) 1 TQN, (**B**) 2V0M, (**C**) 1W0E, and (**D**) 4 NY4.