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.