The binding free energies of both TLR3-MEPVC and TLR4-MEPVC complexes were computed using continuum solvation MMPBSA and its complementary MMGBSA. The binding free energies of the vaccine construct for the receptors were estimated considering molecular mechanics energies as well as solvation energies. The net binding free energy for both complexes revealed to be very high illustrating the high interacting affinity of the molecules. For TLR3-MEPVC, the total free energy of binding is -41.4273 kcal/mol in MMGBSA while in MMPBSA it is -84.4908 kcal/mol (Table 3 ). In case of TLR4-MEPVC, the net binding energy is -13.9690 kcal/mol and -21.2289 kcal/mol in MMGBSA and MMPBSA, respectively. The gas phase energy in TLR3-MEPVC complex is highly dominating and contributes significantly to the overall binding energy. The total gas phase energy of TLR3-MEPVC is -530.5792 kcal/mol in MMGBSA and -530.5792 kcal/mol in MMPBSA. The electrostatic contribution to the net gas phase energy is high i.e. -393.2724 kcal/mol in both MMGBSA and MMPBSA compared to van der Waals energy (-137.3068 kcal/mol). The electrostatic energy contribution to the solvation energy is 507.5596 kcal/mol in MMGBSA and 462.9381 kcal/mol in MMPBSA and is highly non-favorable to the net solvation energy. For TLR4-MEPVC, the net MMPBSA is the comparatively high (-21.2289 kcal/mol) than the MMGBSA (-13.9690 kcal/mol) with solvation energy noticed to dominate the overall interaction energies (Table 4 ). The solvation energy in MMGBSA and MMPBSA is -245.2265 kcal/mol (electrostatic contribution, -240.2983 kcal/mol) and -252.4865 kcal/mol (electrostatic contribution, -248.2779 kcal/mol). The net gas phase energy is 231.2576 kcal/ mol in both MMGBSA and MMPBSA and major favorable contribution is from van der Waals is -35.8526 kcal/mol opposed to the non-favorable contributions is 267.1101 kcal/mol. Residue wise, most of the interacting residues of TLR3 reported in docking assay have very low binding energy reflecting the highly stable nature of these residues in interaction and the key role they are playing in holding the MEPVC at the docked region. The binding energy of the hotspot residues are as follow: His156 (-2.14 kcal/mol), Asp180 (-6.21 kcal/mol), Lys201 (-1.23 kcal/mol), Glu20 (-2.13 kcal/mol), Ser206 (-0.43 kcal/mol), Phe227 (-3.45 kcal/mol), Asp229 (-1.51 kcal/mol), Asp230 (-1.88 kcal/mol), Ser254 (-2.98 kcal/mol), Ser256 (-1.52 kcal/mol), Ser282 (-1.21 kcal/mol), Asp284 (-1.99 kcal/mol), Asp285 (-0.23 kcal/mol), Glu301 (-3.13 kcal/mol), Tyr302 (-1.48 kcal/mol), Phe304 (-3.52 kcal/mol), Glu306 (-3.43 kcal/mol), Tyr307 (-2.89 kcal/mol), Arg325 (-8.12 kcal/mol), His359 (-0.17 kcal/mol), Lys382 (-0.43 kcal/mol), Tyr383 (-3.27 kcal/mol), Pro408 (-2.22 kcal/mol), His410 (-1.16 kcal/mol), Iso411 (-1.81 kcal/mol), His432 (-1.76 kcal/mol), Glu434 (-2.77 kcal/mol), Pro408 (-3.11 kcal/mol), Asp457 (-1.15 kcal/mol), Phe459 (-.5.12 kcal/mol), Gln483 (-4.11 kcal/mol), and Glu533 (-1.23 kcal/mol). In case of TLR4-MEPVC, docking residues Pro23 (-1.54 kcal/mol), Glu24 (-2.31 kcal/mol), Ser25 (-3.33 kcal/mol), Asp44 (-4.43 kcal/mol), Lys47 (-5.23 kcal/mol), Asp50 (-2.21 kcal/mol), Asp51 (-1.99 kcal/mol), Arg67 (-5.65 kcal/mol), Arg87 (-1.21 kcal/mol), Glu89 (-2.11 kcal/mol), Pro113 (-0.12 kcal/mol), Gln115 (-1.21 kcal/mol), Asp137 (-0.23 kcal/mol), His159 (-3.22 kcal/mol), and Asp160 (-2.43 kcal/mol) from chain B and Lys20 (-2,89 kcal/mol), Phe64 (3.21 kcal/mol), and Asp114 (-2.11 kcal/mol) from chain D are hotspot residues.
Table 3 Differences of binding free energy (Complex - Receptor - Ligand) in MMGB/PBSA for the TLR3-MEPVC complex.
Method Net Energy Component Average Std. Dev. Std. Err. of Mean
MMGBSA DWAALS -137.3068 7.0875 0.7088
EEL -393.2724 43.8913 4.3891
EGB 507.5596 41.1568 4.1157
ESURF -18.4077 0.8700 0.0870
DELTA G gas -530.5792 41.8517 4.1852
DELTA G solv 489.1519 41.4134 4.1413
DELTA TOTAL -41.4273 7.7453 0.7745
MMPBSA VDWAALS -137.3068 7.0875 0.7088
EEL -393.2724 43.8913 4.3891
EPB 462.9381 39.2924 3.9292
ENPOLAR -16.8497 0.6609 0.0661
EDISPER 0.0000 0.0000 0.0000
DELTA G gas -530.5792 41.8517 4.1852
DELTA G solv 446.0884 39.5288 3.9529
DELTA TOTAL -84.4908 10.3941 1.0394
Table 4 Differences of binding free energy (Complex - Receptor - Ligand) in MMGB/PBSA for the TLR4-MEPVC complex.
Method Net Energy Component Average Std. Dev. Std. Err. of Mean
MMGBSA DWAALS -35.8526 3.0453 0.3045
EEL 267.1101 29.5270 2.9527
EGB -240.2983 27.5172 2.7517
ESURF -4.9282 0.3849 0.0385
DELTA G gas 231.2576 29.5487 2.9549
DELTA G solv -245.2265 27.4463 2.7446
DELTA TOTAL -13.9690 4.2201 0.4220
MMPBSA VDWAALS -35.8526 3.0453 0.3045
EEL 267.1101 29.5270 2.9527
EPB -248.2779 27.2761 2.7276
ENPOLAR -4.2085 0.2122 0.0212
EDISPER 0.0000 0.0000 0.0000
DELTA G gas 231.2576 29.5487 2.9549
DELTA G solv -252.4865 27.2706 2.7271
DELTA TOTAL -21.2289 5.3805 0.5380