The spike protein (S) of SARS-coronavirus (SARS-CoV) interacts to its cellular receptor, Angiotensin Converting Enzyme 2 (ACE2) via Receptor Binding Motif (RBM; aa 424–494) present in receptor binding domain (RBD). 18 residues of the receptor make contact with 14 residues of the viral spike protein, mainly by hydrophilic interactions. Tyrosine residues are present at this interface, in RBM . Besides these, Prabakaran et al.  also noted the importance of tyrosines while studying the binding interface of the RBD and neutralizing antibody. This tyrosine was also noted by Prabakaran et al.  as an important residue in making interaction to neutralizing antibody. We also observed important role of tyrosines; the phytocompound, ‘Withanone’, was well-bound at the ACE2-RBD interface by two H-bonds (Tyr16 of ACE2 and Tyr175 of RBD to Withanone) (Table 1, Fig. 2), and upon mutation of RBD Tyr175 to Ala, the phytocompound preferred different location.
MD simulation of ACE2-RBD complex was performed using NAMD, with or without the ligand molecule (Withanone). The Withanone moved slightly towards the centre of the binding interfacewith a formation of three new H-bonds (ACE2 N15, ACE2 Q19 and RBD R78 to Withanone) (Figs. 3A and 3B). Further, we explored ionic interactions at the binding interface of the modeled ACE2 receptor and RBD of 2019-nCoV.Two inter-chain (binding interface) salt bridge interactions were detected: Glu12 OE2– Lys87 NZ (2.75 Å) (aa 404) and Glu20 OE2 – Arg73 NZ (2.67 Å) (aa 390) (Fig. 4A). These salt bridge interactions play an important role in stabilization of the ACE2 receptor and RBD complex. Val residue (aa 404) in SARS-CoV is substituted by Lys in COVID-19 S protein RBD, and Lys (aa390) is substituted by Arg. Overall, the increase in salt bridge number in the receptor-RBD binding interface makes the complex more stabilized in COVID-19 as compared to SARS-CoV (Fig. 4A). Percent occupancies of the salt bridges were decreased in the simulation trajectories with the Withanone, as compared to the trajectories without the Withanone. Overall, there were observed effects of the ligand incorporation in these salt bridges occupancies. Longer simulation is needed to observe whether these salt bridges are completely broken by incorporating the Withanone.
Protein surfaces have many hydrophilic residues, and salt bridges present in the surface play an important role in protein-protein association or binding . Hence the protein interface (binding interface) is generally more hydrophilic than the protein interiors. Xu et al.  showed that electrostatic interactions play an important role in protein binding than in folding. Hence the interfacial salt bridges, which are the major contributors to the electrostatic interactions between proteins, get central role in binding events. Generally, the structures of the proteins do not change significantly upon complex formation, but some conformational rearrangements are observed, and most of these are in side chain movements . Geometrical complementarity and stability in energetic are the two factors to occur for protein binding, and the hydrophobic effect, hydrogen bonds and salt bridges are the key players in energetic. A salt bridge can provide favorable free energy to the binding , on the other hand, an isolated charge without forming a salt bridge, when buried in the protein interface, could substantially destabilize binding, due to the to the desolvation cost. We calculated energetics of the two salt bridges (E12-K87 and E20-R73) at the interface of ACE2 and RBD. Both the salt bridges were stabilizing initially (-1.36 kcal/mol and − 6.15 kcal/mol, respectively), but turned into destabilizing (salt bridge E12-K87, 1.53 kcal/mol) or less stabilizing (salt bridge E20-R73, -2.46 kcal/mol) as seen in last frame of the MD simulation (Fig. 5). These results clearly indicated that the incorporation of the Withanone had pronounced effect on the salt bridges and on protein complex stability.
In the current work, we have presented the results of a MD simulation of the ACE2-RBD complex with and without Withanone. The data shows the local RMSD changes, a measure of flexibility, of non-H atoms of the binding interface residues of RBD (aa 424 to 494) (Fig. 6). RMSD was decreased in the simulation of Withanone plus case, in the region of aa424 to 494. It may be due to the involvement of these contact residues in electrostatic interactions with the ligand.
The electrostatic component of the binding free energies of ACE2-RBD complex were estimated on 11 trajectories (simulated without the ligand) and 10 trajectories (simulated with the ligand), to assess the hypothesis that the proposed phytocompound weakens the interactions between ACE2 and RBD. ΔΔGel was decreased by 0.6 kcal/mol in the simulation trajectories with the Withanone, compared to the trajectories simulated without the Withanone (8.89 and 8.33 kcal/mol in the trajectories without or with the ligand, respectively) (Figure. 7). Whereas, the ΔΔGel of the complex with the ligand (7.27 kcal/mol) was decreased by 4.3 kcal/mol as compared to that without the ligand (11.55 kcal/mol). Such a decrease in electrostatic component of binding free energy clearly indicates that the binding of Withanone at the interface of the ACE2 and RBD weakens their interactions.