6.0 Binding Pocket Identification of AKHRs, Molecular Docking Calculations, and MD
6.4.2 Accessibility of the AKHRs binding pocket, and determination of binding mode during
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region as the best binding site and ranked 1, and the ECL2 does not cover the binding site. The best explanation for this is the receptor is inactive. The ranked extracellular region as 1 is expected because the location of the binding cavity and the participation of the entire extracellular domain conform with experimental data239,441. The same situation of ligand binding to the extracellular domain was reported for the human gonadotropin-releasing hormone receptor 442.
Having obtained the position of the preferred binding sites for the AKHRs, more focused docking was carried out to determine the binding mode of the AKHRs.
6.4.2 Accessibility of the AKHRs binding pocket, and determination of
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Figure 6. 3. The binding of Phote-HrTH to all three β2AR-based AKHRs. * a) flf = The flesh fly AKHR b) frf = The fruit fly AKHR c) off = oriental fruit fly AKHR. The colours represent H1-H6.
In contrast, in Rhodopsin models, even when the ligand was forced into the identified binding site from the extracellular region, the ECL2 had already covered the binding site leaving a tiny space. Unfortunately, the available space between the loops and the helices was too small for the ligand to access the binding cavity. Also, for the Rhodopsin models, when the Glide S- peptide docking command was initiated, no poses were generated, indicating the absence of cavities. While in the β2AR-based AKHRs, because of their open conformation, it was easy for proper diffusion of the peptide in and out of the binding site access. Since the Rhodopsin AKHR models are closed and would not allow further probing, by allowing the peptide to sink in, one could postulate that the β2AR-models could represent the conformation of the receptor/inactive AKHR. At the same time, the Rhodopsin-model is used as a control. Hence, provide information on the active AKHR state of each receptor.
6.4.2.1 Molecular docking calculations for the β2flf-AKHR
Crucial interactions between Phote-HrTH peptide and β2flf AKHR were observed after the S- peptide docking. The docking scores were -13.20 kJmol-1, and the ΔGbind (MMGBSA) was -
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75 kJmol-1. The most critical interaction is a salt bridge experienced between the Asp7 of Phote- HrTH and Arg303 of the receptor. To provide a significant amount of binding enthalpy and stability to the system, a - stacking interaction was experienced between Trp8 of Phote- HrTH and His 203 of the ECL2. This interaction with ECL2 help closes the loop over the binding site. These observations were similar to the studies on cation-pi interaction reported by Dougherty 379,443. The ligand interaction maps are presented in Figure 6.4. The -cationic interaction falls at 4.64Å, which is within the confines of < 6 Å proposed by Gallivan and Dougherty 380.
Figure 6.4: Ligand interaction diagram of Phote-HrTH bound to the β2flf model of Sarcr-AKHR. Residues are
represented as coloured spheres, labelled with the residue name and residue number, and coloured according to their properties, green for hydrophobic, blue for positive charge and red for a negative charge. The ligand is displayed as a 2D structure. Interactions between the residues and the ligand are drawn as lines, coloured by interaction type, purple for H-bonding and green for pi-pi stacking. The binding pocket is indicated by a line drawn around the ligand, in the
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same colour as the nearest residue. Solvent exposure is indicated on the ligand atoms, and by the break in the line drawn around the pocket.
6.4.2.2 Molecular docking calculations for the β2frf-AKHR
A β2frf putative binding site with Phote-HrTH is similar to those reported for Anopheles gambiae by Jackson et al., 324. The docking scores were -14.33 kJ mol-1, and the ΔGbind
(MMGBSA) was -79 kJ mol-1. The most critical interaction is a salt bridge between Asp7 of Phote-HrTH and Arg301. There is H-bonding between Phote-HrTH Ser5 and Val103 of ECL1 and Trp99 of TM2. The NH of Phote-HrTH Ser5 and C=O Asp7 H-bond to Ser196 on ECL2.
Also, Phote-HrTH Trp8 NH interacts with Asp197 of ECL2. These interactions with ECL2 are interesting as this loop closes over the binding site after the ligand binds. The terminal amide of ligand H-bonds to Ile296 of ECL3. To provide proper stability and a significant amount of binding enthalpy to the system, -stacking interactions were experienced between Trp8 and Phe4 of Phote-HrTH and Phe198 of ECL2. Hence, the - stacking interaction of 3.94 Å was noticed, which falls within the confines of < 6 Å as proposed by Gallivan and Dougherty 380 The ligand interaction is presented in Figure 6.5.
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Figure 6. 5: Ligand interaction diagram of Phote-HrTH bound to the β2frf model of Drome-AKHR. Residues are represented as coloured spheres, labelled with the residue name and residue number, and coloured according to their properties, green for hydrophobic, blue for positive charge and red for a negative charge. The ligand is displayed as a 2D structure. Interactions between the residues and the ligand are drawn as lines, coloured by interaction type, purple for H-bonding and green for pi-pi stacking. The binding pocket is indicated by a line drawn around the ligand, in the same colour as the nearest residue. Solvent exposure is indicated on the ligand atoms, and by the break in the line drawn around the pocket.
6.4.2.3 Molecular docking calculations for the β2off-AKHR
β2off putative binding site with Phote-HrTH is similar to those reported for Anopheles gambiae by Jackson et al., 324, frf-model and flf-model. The docking scores were -14.03 kJmol-1, and the
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ΔGbind (MMGBSA) was -82 kJmol-1. Lys 306 was also discovered to act as an anchor in the binding pocket, forming a π-cation and hydrogen bond interaction with Phe4 of Phote-HrTH.
A π-cation interaction of 4.96 Å was observed, which falls within the confines of < 6 Å as proposed by Gallivan and Dougherty. Also, a salt bridge was identified between Arg 306 and Asp 7 Phote-HrTH (see Figure 6.6). These interactions are similar to those shown for β2frf and β2flf.
Figure 6. 6: Ligand interaction diagram of Phote-HrTH bound to the β2off model of Bacdo-AKHR. Residues are represented as coloured spheres, labelled with the residue name and residue number, and coloured according to their properties, green for hydrophobic, blue for positive charge and red for a negative charge. The ligand is displayed as a 2D structure. Interactions between the residues and the ligand are drawn as lines, coloured by interaction type, purple for H-bonding and green for pi-pi stacking. The binding pocket is indicated by a line drawn around the ligand, in the same colour as the nearest residue. Solvent exposure is indicated on the ligand atoms, and by the break in the line drawn around the pocket.
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The ligand interaction diagram is comparable to one another since they all have hydrogen interactions, salt bridge, ionic interactions and - stacking interactions, though β2frf has two
- stacking interactions originating from same molecule Phe4 and Trp8 of Phote-HrTH and
Phe198 of the receptor, while β2flf has a - stacking interactions between Trp8 of Phote- HrTH and His203 and β2off possess a π-cationic interaction between Lys 306 and Phe4. The
- stacking and π-cationic interactions experienced by these AKHRs and the ligand signifies discrepancies in the strength at which the molecules bind.