Trajectory analysis
The dynamics stability of the RBD-ACE2-naltrexone complex was analyzed by performing all-atoms MD simulations of 100 ns 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 Å throughout the entire simulation (Figure 3(A)). Besides, the RMSD of ligand was also found to be stable (red line in Figure 3(A)) 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 3(B)). The residue flexibility of protease and RBD-ACE2/Naltrexone complex was examined by performing Cα RMSF analysis of both the sub-units (Figure 3(C)). The average RMSF of ACE2 was found to be 0.14 nm, 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 process (Dehury et al., 2014). 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 4(A)). 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 to 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 the complex retained its structural integrity throughout the simulation (Figure 4(B)). 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 4(C)) 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.
Figure 4.  Inter-molecular hydrogen bond dynamics and structural superposition of the initial complex with the simulated RBD-ACE2-naltrexone complex during 100 ns MD. (A) Dynamics stability of RBD-ACE2-naltrexone complex with respect to inter-molecular hydrogen bonds along the 100 ns time scale. (B) Structural superimposed view of the starting complex used for MD (green) and the snapshot obtained from clustering analysis (cyan) of MD trajectory during the last 50 ns. (C) Inter-molecular contacts of the docked complex and MD simulated complex.