3.8.1 MM/GBSA free energy calculations
To understand the thermodynamics behind the protein-DNA complexes, MM-GBSA was calculated to determine the binding mechanisms. The calculations were performed to calculate the binding energies of WT protein comparing it to mutant protein in the DNA-protein complex. Based on the 200ns MD trajectories of WT and variants the energy analysis and its corresponding component as calculated and reported in Table 3. The results indicated high binding energy (-216.33 Kcal/mol) compared to most other variants. The point mutations decreased the positive polar term resulting in overall increase of the negative term promoting DNA-protein complex formation. The energy components included here are Van der Waals, electrostatic interaction, non-polar and polar energies contributing to the total binding energy and favouring complex formation. Primarily, the stability of the complex was majorly due to the electrostatic interaction energy whereas other energy terms contributed very less to the total energy of the complex. The spontaneity of this interactions is highlighted by the high negative value of the electrostatic interactions, whilst polar solvation energies hinders the interactions between DNA and protein. Interestingly, some of the residues showed higher binding stability when mutated resulting in higher negative values in T144R, A149V and Q188E. N-terminal R (arginine) showed a loss in interaction energy (-188.74 Kcal/mol) when mutated to lysine. However, when mutated to tryptophan, it showed considerable increase in binding energy (-224.41) of the mutant. Studies have highlighted the role of tryptophan in DNA binding domain to be important, which might be the case in this mutant also76. The same effect was observed on the C-terminal R when mutated to tryptophan (R198W= -224.43 Kcal/mol). But when mutated to cysteine (-186.36 Kcal/mol) and histidine (-185.16 Kcal/mol) there was a loss of interaction energy of the mutant. These residues were also found to be highly conserved in the Consurf analysis. Furthermore, this also implies about the role of these conserved residues to predominantly be the fundamental residues driving a compact interaction with DNA.