3.2.3.  Essential residues for polyphenols binding
Further, to gain a deeper insight into the best four RdRp/polyphenols and remdesivir interaction pattern, the total binding free energy was decomposed into polyphenols-residue pair based on the MM-GBSA scheme. The approach of per-residue based contributions is useful to determine the binding mechanisms of an inhibitor at an atomistic level, and it also reveals the individual residue contributions. The different energy contributions from the backbone and side-chain of each residue are shown in Figure 5 and listed in Table 5.
Figure 5.  Decomposition of the binding free energy into contributions from individual residues for RdRp complexed with remdesivir, EGCG, TF3, TF2b and TF2a.
Table 5.  Per-residue based decomposition of binding free energy for the complex of remdesivir, EGCG, TF3, TF2a and TF2b with the SARS-CoV-2 RdRp.
Residue  TvdW  Telec  Tpol  Tnp  Tside_chain  Tbackbone  Ttotal
RdRp/Remdesivir
Asp761  1.05  −21.73  18.81  −0.14  −1.82  −0.19  −2.01
Lys798  −2.74  −4.28  5.74  −0.47  −1.76  0.01  −1.75
Pro620  −1.42  −1.15  1.16  −0.18  −1.26  −0.33  −1.59
Asp760  −0.57  −4.27  4.24  −0.12  −0.20  −0.52  −0.72
Arg553  −2.07  −3.71  5.61  −0.51  −0.76  0.08  −0.68
RdRp/EGCG
Asp452  1.65  −16.48  9.85  −0.08  −5.15  0.09  −5.06
Arg553  −3.83  −4.84  6.59  −0.59  −2.68  0.01  −2.67
Pro620  −1.64  −0.41  0.51  −0.33  −1.62  −0.25  −1.87
Asp618  0.49  −8.14  6.66  −0.09  −1.13  −0.05  −1.08
Lys621  −2.26  −5.12  6.92  −0.50  −0.87  −0.09  −0.96
RdRp/TF3
Asp761  2.52  −30.13  22.11  −0.25  −5.54  −0.21  −5.75
Arg836  −0.83  −14.95  12.29  −0.37  −3.85  −0.01  −3.86
Arg555  −5.80  −4.00  7.29  −0.70  −2.98  −0.23  −3.21
Thr556  −0.20  −3.96  2.44  −0.13  −0.21  −1.64  −1.85
Ile548  −1.03  −0.08  0.12  −0.13  −0.65  −0.47  −1.12
Ser814  −1.67  0.09  0.78  −0.08  −0.48  −0.40  −0.88
Val557  −0.65  −0.16  0.17  −0.24  −0.65  −0.23  −0.88
RdRp/TF2b
His816  −3.00  −0.81  1.69  −0.32  −1.63  −0.81  −2.44
Asp833  −1.29  −0.23  0.46  −0.18  −0.21  −1.03  −1.24
Tyr877  −0.92  −2.06  2.06  −0.27  −0.43  −0.76  −1.19
Glu811  0.22  −9.24  8.11  −0.13  −2.07  1.03  −1.04
His810  −1.88  −0.41  1.66  −0.34  −0.10  −0.85  −0.95
Tyr831  −1.51  −0.36  1.19  −0.13  −0.21  −0.60  −0.81
Asn815  −0.60  −0.27  0.09  −0.01  −0.25  −0.54  −0.79
RdRp/TF2a
Asp618  2.63  −20.71  13.40  −0.17  −4.84  −0.01  −4.85
Arg553  −3.80  −6.52  7.55  −0.69  −2.68  −0.78  −3.46
Lys551  −2.23  −0.80  2.23  −0.45  −0.84  −0.41  −1.25
Arg555  −1.33  −0.62  1.05  −0.27  −1.12  −0.05  −1.17
Glu167  0.42  −6.82  5.92  −0.12  −0.61  0.01  −0.60 As shown in Figure 5, it was observed that residues favoring the binding of the polyphenols with RdRp include Asp452, Arg553, Arg555, Val557, Asp618, Pro620, Lys621, Asp623, Arg624, Asp760, Asp761, and Glu811, Asp833, and Arg836. Most of these residues are located in the binding site of RdRp and can form direct contacts with polyphenols and remdesivir. Figure 5 shows that amino acids Pro620, Asp761 and Lys798 for RdRp/remdesivir; Asp452, Arg553, Pro620 and Lys621 for RdRp/EGCG; Ile548, Arg555, Thr556, Asp761 and Arg836 for RdRp/TF3; Glu811, His816, Asp833 and Tyr877 for RdRp/TF2b; Lys551, Arg553, Arg555 and Asp618 for RdRp/TF2a contributed more favorably towards the binding by contributing more than −1.0 kcal/mol in size.
To complement the energetic analysis, we performed MD trajectory-based hydrogen bond (h-bond) analysis for all five complexes, and the h-bonds with occupancy are listed in Table 6. The h-bonds were determined by setting the acceptor-donor distance of ≤ 3.5 Å, and the angle cut off ≥ 1200. Important h-bonds between RdRp-inhibitors are shown in Figure 6. In the case of RdRp/remdesivir, key residues involved in the hydrogen bonding are Asp761, Asp760, and Ser759, respectively. Asp760 is found to form two h-bonds with remdesivir (Asp760@OD2 - Lig@O7, Asp760@OD2 - Lig@O6) with an occupancy of more than 15% (see Table 6 and Figure 6). In the case of RdRp/EGCG, both Asp618 and Asp760 form two h-bonds with the ligand with an occupancy in the range of 16.09 to 30.17%. On the other hand, Asp761 form an h-bond with TF3 (Asp761@OD1 - Lig@O11) with an occupancy of 69.84%, while Arg836 forms two h-bonds with the ligand (Arg836@NH2 - Lig@O14, Arg836@NE - Lig@O14) with an occupancy of 52.66%, and 48.70%, respectively. Glu811, Thr556 and Asp761 also formed h-bonds with the ligand during our simulations with an occupancy varying in the range of 44% to 58% (see Table 6). In the case of RdRp/TF2b, Glu811 is found to form two strong h–bonds with the ligand (Glu811@OE1 – Lig@O7, Glu811@OE2 – Lig@O7) with an occupancy of 22.45% and 18.89%, respectively. On the other hand, it can be observed from Table 6 that Pro832 and Tyr877 form strong h-bonds (Pro832@O -Lig@O8 and Lig@O10 -Tyr877@OH) with increased occupancy (> 24%). Finally, in the case of RdRp/TF2a, Asp618 is found to form two strong h-bonds with the TF2a (Asp618@OD1 – Lig@O10, Asp618@OD1 – Lig@O11) with an occupancy of 38.68% and 38.38%, respectively. Asp760 also forms a h-bond (Asp760@O – Lig@O11) with an occupancy of 20.83%.
Figure 6.  Five main hydrogen bond interactions between ligands and RdRp.
Table 6.  Main hydrogen bond interactions formed by RdRp with remdesivir and polyphenols along with the corresponding average distance and percentage of occupancy determined using the trajectories of production simulations.
Acceptor  Donor  Avg. Distance (Å)  Occupancy (%)
RdRp/Remdesivir
Asp760@OD2  Lig@O7  2.66  19.46
Asp761@OD1  Lig@O7  2.65  17.70
Asp761@OD2  Lig@O6  2.63  16.86
Asp760@OD2  Lig@O6  2.65  16.65
Lig@O6  Ser759@OG  2.80  11.63
Asp760@OD1  Lig@O7  2.66  10.59
RdRp/EGCG
Asp618@OD1  Lig@O5  2.61  30.13
Asp618@OD1  Lig@O6  2.61  29.28
Asp618@OD2  Lig@O5  2.61  18.25
Asp618@OD2  Lig@O6  2.61  17.38
Asp760@OD1  Lig@O5  2.63  16.09
Tyr455@OH  Lig@O11  2.83  10.97
RdRp/TF3
Asp761@OD1  Lig@O11  2.61  69.84
Glu811@O  Lig@O10  2.76  58.43
Thr556@O  Lig@O3  2.72  56.95
Lig@O14  Arg836@NH2  2.83  52.66
Lig@O14  Arg836@NE  2.86  48.70
Asp761@OD2  Lig@O20  2.62  44.51
RdRp/TF2b
Pro832@O  Lig@O8  2.77  26.31
Lig@O11  Tyr877@OH  2.75  24.39
Glu811@OE1  Lig@O7  2.65  22.45
Glu811@OE2  Lig@O7  2.65  18.89
Asp833@OD2  Lig@O8  2.65  12.01
Asn874@OD1  Lig@O11  2.68  8.58
RdRp/TF2a
Asp618@OD1  Lig@O10  2.59  38.68
Asp618@OD1  Lig@O11  2.62  38.38
Asp760@O  Lig@O11  2.70  20.83
Asp618@OD2  Lig@O11  2.62  16.59
Asp618@OD2  Lig@O10  2.58  16.49
Asp618@OD1  Lig@O15  2.67  16.37 Finally, we supplemented the above results by analyzing the final conformation of each production simulation with the help of 2D LigPlot+ software, and different h-bonds and hydrophobic interactions were shown in Figure 7. Hydrogen bonds are depicted in green dotted lines, while red semicircle residues are involved in hydrophobic interactions. For the RdRp/remdesivir complex, Figure 7(A) displayed nine hydrophobic interactions with Lys545, Ala547, Ser549, Arg553, Val557, Asp684, Ser759, Ser814, and Arg836. This large number of interactions account for the high stability and good binding affinity of remdesivir to RdRp. EGCG formed hydrophobic interactions with Lys551, Ala554, Arg553, Arg624, Pro620, (Figure 7(B)). In the case of TF3, eight hydrophobic interactions with His439, Ile548, Ser814, Phe812, Val557, Ser549, Tyr619 and Arg555 were formed as revealed by Figure 7(C). Figure 7(D) shows that seven hydrophobic interactions with Asp833, His816, Pro832, Gln815, His872, His810 and Ser434 were formed for RdRp/TF2b. Finally, Figure 7(E) shows that RdRp/TF2a formed hydrophobic interactions with Arg555, Ala554 and Lys551. Overall, TF3 has a higher binding affinity toward RdRp compared to the other polyphenols due to a larger number of stable hydrogen bonds and hydrophobic interactions.
Figure 7.  The RdRp-ligands interaction profile for (A) RdRp/remdesivir, (B) RdRp/EGCG, (C) RdRp/TF3, (D) RdRp/TF2b and (E) RdRp/TF2a. The polyphenols and remdesivir are shown in balls and sticks. Hydrogen bonds are depicted in green dotted lines, and red semicircles residues are involved in hydrophobic interactions.