*2.3. Molecular Dynamics Simulation*

The best docked location of HPLD and CTtD with the highest docking score at 100 ns molecular dynamics (MD) generated by AutoDock Vina was used. Only the best docking

output was used to build up this method in a high-throughput manner for analyzing the binding mechanism of the ligand at the active site of protein under clearly defined aqueous conditions. The root mean square deviation was computed to determine structural stability from MD data (RMSD). HPLD and CTtD formed stable conformation after ~80 ns and ~65 ns, respectively, with an appropriate RMSD value of 3.04 and 2.41 Å, respectively, as seen in the RMSD plot (Figure 9).

The RMSD value range of <3.0 Å is the most acceptable [17]. CTtD produces a more stable combination as a result of this discovery. The MD findings of ligand–receptor interaction, as shown in Figure 10, bring protein chains closer together and close the distance between them [18]. Chimera 1.15 software was used to create the superimposed structures by employing the tool–structure comparison followed by the MatchMaker feature. Pairing uses both sequence and secondary structure to superimpose comparable structures.

**Figure 10.** Superimposed structures of (**a**) unbounded dopamine receptor (orange) and dopamine receptor after simulation (blue) for HPLD and (**b**) unbounded dopamine receptor (orange) and dopamine receptor after simulation (pink) for CTtD.

RR Distance Maps creates a distance map through Chimera 1.15 software by a structural comparison tool. The map can display Cα-Cα distances within a single protein chain, as well as averages and standard deviations for many chains. The white diagonal on the map represents no distance between the two residues, but the red and blue on the map reflect residue pairings with the biggest distance differences between the two conformations (Figure 11).

**Figure 11.** RR distance map: (**a**) unbounded dopamine receptor and dopamine receptor after simulation for HPLD and (**b**) unbounded dopamine receptor and dopamine receptor after simulation for CTtD, [PAS (#1) value = protein after simulation, SD = Standard deviation].

The average radius of gyration (Rg) values for HPLD and CTtD were 22.647 and 22.586 Å, respectively. Along the simulation time, Rg decreased, indicating that the structures became more compact (Figure 12).

**Figure 12.** Radius of gyration for HPLD (black) and CTtD complexes (blue) during the 100-ns simulation time.

The number of hydrogen bond interactions between HPLD and CTtD was displayed against time using a grid-search on a 27 × 18 × 21 grid with rcut = 0.35 (Figure 13). There were 396 atoms of donors and 753 atoms of acceptors detected when the hydrogen bonds were between the ligand at 36 and 53 atoms for HPL and CT complex, respectively, and 2941 atoms of the dopamine receptor were calculated. Out of a total of 201,657 potential bonds, the average number of hydrogen bonds per period for HPLD and CTtD was 1.738 and 2.481, respectively.

Overall, the receptor–protein interaction enhanced the number of hydrogen bonds substantially, and it was more in CTtD.

The values of solvent accessible surface area (SASA) altered as the ligand bound to the receptor (Figure 14). When the receptor binds to a ligand, the SASA value drops, indicating a change in conformation in the protein structure and a smaller pocket with more hydrophobicity around it.

**Figure 13.** Number of average hydrogen bonding interactions between (left) HPLD complex and (right) **CTtD** complex during the 100 ns simulation time.

**Figure 14.** Solvent-accessible surface area analysis for HPLD complex (black) and CTtD complex (blue) during the 100 ns simulation time.
