3.5.1. RMSD Analysis of Protein and Protein–Ligand Complexes

The RMSD patterns for C-alpha atoms of NEK7 protein were estimated in order to determine the effect of the bounded drug on the conformational stability of NEK7 protein. Figure 11 is displaying the progression of RMSD values for the C-alpha atoms of NEK7 as a function of time. The 2wqn–Dabrafenib complex reaches the equilibrium after around 5 nanoseconds of simulation, and although side chain residues displayed fluctuations, they remained in the permissible range of 1–4 angstroms, which can be considered insignificant [60]. The NEK7–Dabrafenib complex showed slight fluctuations after 50 ns, which again became stable after 60 ns of simulation and remained equilibrated throughout the simulated trajectory. RMSD fluctuation was observed from 70 to 90 ns, which is due to the decrease in the number of contacts during this time, but after 90 ns, the number of contacts with amino acid residues increased and RMSD pattern became stable. It demonstrate the existence of stable molecular interactions. After being equilibrated, NEK7 RMSD values fluctuated within 2 angstrom. After 80 ns, protein RMSD showed slight fluctuation up to 2.5 angstrom and dropped again after 95. The average RMSD value for the protein–ligand complex and NEK7 protein is tabulated in Table 5.



These findings suggest that the ligands stayed firmly bound to the receptor throughout the simulation period. Moreover, small RMSD patterns indicate the fewer structural rearrangements and lesser conformational changes within binding site residues [61].

It is beneficial to identify local differences in the protein chain by using the root-meansquare fluctuation (RMSF). Figure 12 peaks on the RMSF graph represent the regions of the protein that change the most during the simulation. The average RMSF for the NEK7 backbone was 0.87 angstrom, indicating the fewer structural rearrangements (Table 5). The N- and C-terminal ends of proteins are more likely to undergo alteration than any other portion of the protein. In the range of amino acids from 180 to 220, the RMSF value fluctuated, as can be seen in the RMSF graph. These residues are found in the C-terminal lobe. The protein's structure, such as its alpha and beta helices and strands, tends to be stiffer and less variable than its unstructured component. According to MD trajectories, the

residues with the highest peaks are found in loop areas or the N- and C-terminal regions. Binding site residues with low RMSF values imply a stable ligand–protein interaction.

**Figure 11.** Residue wise root-mean-square deviation (RMSD) of the C-alpha atoms of NEK7 (2wqn) and Dabrafenib Complex.

**Figure 12.** Root-mean-square fluctuations (RMSF) of the C-alpha atoms of NEK7 (2wqn).

The contact profiles of NEK7–Dabrafenib were computed from simulated trajectories, as shown in Figure 13. FDA-approved drug Dabrafenib interacted with ILE40, LYS163 and

ARG121 through Water Bridge and hydrogen bonding. The amino acid residues, ILE40, LYS163, and ARG42, were involved in H-bonding. During MD simulations, 12 hydrogen bonds were found to be dominant with significant occupancy. Details of hydrogen bonding is given in Table 6.

**Figure 13.** NEK7-Dabrafenib Contact histogram.




**Table 6.** *Cont.*

The hydrogen bond with ALA114 existed for more than 25% of simulation time. Hydrophobic interactions existed between VAL48, LEU113, VAL48, and PHE168. These molecular interactions contributed towards stabilizing the protein–ligand complex.
