Trajectory analysis.
The dynamics stability of the RBD-ACE2-naltrexone complex was analyzed by performing all-atoms MD simulations of 100ns in GROMACS. The backbone RMSD analysis provides important information on the stability of protein and protein-ligand complexes and the time when simulation reached equilibrium. The RMSD of the RBD-ACE2-naltrexone complex displayed an average RMSD ~2.46 nm throughout the entire simulation (Figure 3A ). Besides, the RMSD of ligand was also found to be stable with very minimal deviation as compared to the starting conformation. Overall, the complex system displayed the least backbone deviation, indicates that docked conformation is accurate and remained stable over the 100 ns timescale. Radiuses of gyration assess the compactness of the system, where a compact gyradius of ~3.24 nm for the complex indicates the consistent shape and size of the system during the simulation (Figure 3B ). The residue flexibility of protease and RBD-ACE2 structures were examined by performing Cα RMSF analysis of both the sub-units (Figure ). The average RMSF of ACE2 was found to be 0.14nm, while for the RBD it was reported to be 0.17 nm (for the receptor-binding motif ~0.16 nm). The receptor-binding motif of RBD displayed a high degree of flexibility and the residues participated in the ligand interaction also portrayed higher RMSF indicating their participation in ligand recognition.
The intermolecular hydrogen bonds (H-bonds) between interacting atom pairs in a protein-ligand complex plays a vital role in the stability and molecular recognition process17. The intermolecular H-bonds were calculated with respect to time during the 100 ns MD simulation to see the dynamics stability RBD-ACE2-naltrexone complex (Figure 4A ). Though we observed an increased differential H-bonding during the initial 20 ns equilibration phase, however a stable trend with an average of ~4.13 H-bonds are noticed from 60-100 ns. Close inspection of snapshots from MD revealed that some of the H-bonds were broken out during MD simulation, but at a later stage they well rewarded by new H-bonds, and hydrophobic contacts. This may be due to the structural re-orientation of ligand naltrexone in the binding pocket. The structural superposition of the docked complex with the cluster representative obtained from clustering analysis displayed Cα RMSD of 0.65 Å indicated that complex retained its structural integrity throughout the simulation (Figure 4B ). However, close observation of the ligand for the initial starting structure used MD revealed that the ligand tends to reorient within the binding site during MD (as shown in Figure 3C) but form a close tight network of hydrogen bonds and non-bonded contact with ACE and RBM of RBD. Analysis of the cluster representative revealed the crucial residues of RBD and ACE2 involved in the crucial interaction with naltrexone. Lys417 and Asp405 from RBD formed two hydrogen bonds with naltrexone, while Glu37 of ACE2-formed the lone hydrogen bond(Figure S2 ). Many electrostatic and hydrophobic contacts were also observed in the complex (Figure S2 ) where, Ile418, Gln409, and Tyr505 from RBD consistently formed close contact with ligand indicates their strong participation in the interaction mediated by naltrexone.