Analysis of Molecular Dynamics of RBD-ACE2 complex
All-atom aMD simulations allowed to explore the conformations of protein-protein complex over time for each system: RBDWT-ACE2, RBDalpha-ACE2, RBDOmicron-ACE2 and RBDDelta-ACE2 complex. Figure 1 shows the RMSD during 200 ns of aMD for each system with respect to the reference structure of the equilibrium step. RBDWT-ACE2, RBDalpha-ACE2 and and RBDDelta-ACE2 complexes were within fluctuation in a range of 1 to 3 Å (Figure 1), while the RBDOmicron-ACE2 complex the present the different variation during simulation in a range of 1 to 4 Å (Figure 1). Therefore, the structural equilibrium was reached for all system (Figure 1).
In order to obtain insight into flexibility of each residue in protein-protein complex, we have analyzed the Root-Mean-Square Fluctuations (RMSF) taken into consideration the fluctuations of the backbone atoms. In the RMSF analysis (Figure 2) ACE2 shows the greatest fluctuation in regions 123 to 178 (in magenta), 395 to 425 (in red) and in the region of residues 248 to 368 (in yellow), that moves to interact with the viral RBD. the RBDalpha residues show less fluctuation compared to the WT and its last variants (Delta and Omicron).
In this study, we also explore the flexible region in protein-protein complex. through essential dynamics analysis. The PCA graphs, were obtained using the combinations of PC1vsPC2, PC2vsPC3 and PC3vsPC1 (Figure S2), in which the clusters demonstrate two possible states for all systems in PC1vsPC2. The color scales represent the trajectory time of the MD, separating the beginning of the structures in the initial time of the final structures of the MD, however, the Alpha variant already has a greater number of clusters, where each time interval is separated into small clusters.
For Omicron system the structures are visibly separated into blue structures and red structures (see SI, Figure S2), indicating that the initial structures differ from the final ones, leading to variations in the aMD structures (Figure 3). The PCA analysis showed that the RBDWT and the RBDomicron variant present greater conformational fluctuations, however, the RBDalpha variant stands out for its greater stability. In PC1 there are not many movements in RBD and ACE2 (Figure 3). The main movement of RBDWT and RBDomicron is similar because they have a greater number of movements. The Spike protein, via RBD, when it binds, causes changes in ACE2, as shown in Figure 3. The other conformational changes are shown in PC2 and PC3 in Figure S3 for all systems.
Binding Free Energy MMGBSA and Decomposition by Residue
To assess the affinity of the virus for the human receptor and a possible potential risk of immune evasion by the variants, we calculated the free energy using MM/GBSA (∆Gbind (MMGBSA)) based on the points of greatest stability of the aMD trajectory (see Table 1). The RBDomicron shows the highest binding affinity to ACE2, reflecting the infectivity process, but its conformational fluctuations is similar to the other variants. RBDomicron present an adaptive and non-aggressive process when compared to the RBDalpha (with free energy of binding equal to -62.7836 kcal/mol), which demonstrated the lower free energy than RBDWT (-59.7205 kcal/mol). Based on the higher conformational stability of the Alpha variant the high risk is evident and demonstrates a worrying risk of immune evasion due to its degrees of affinity with ACE2.
The RBDDelta has a higher binding affinity with the human receptor compared to the RBDWT (-66.1357 kcal/mol), which demonstrates the great concern of infections based on this variant. The high risk of infectivity is pointed out as greater among the variants because they have a more favorable ∆Gbind in comparison to RBDWT. Therefore, the risk of evolution and emergence of new variants may represent a major health concern due to the degree of affinity that evolves the greater affinity for the human receptor.
Table 1: Binding free energy for native systems (SARS-CoV-2) and variants (Alpha/Delta/Omicron).
Energy (kcal/mol)
|
WT
|
Alpha
|
Delta
|
Omicron
|
∆Evdw
|
-95.6(0.18)
|
-107.3(0.21)
|
-103.4(0.16)
|
-96.4(0.16)
|
∆Eele
|
-625.8(0.94)
|
-608.5(0.91)
|
-955.1(1.05)
|
-1381.7(1.24)
|
∆EGB
|
675.0(0.87)
|
667.5(0.87)
|
1006.3(1.01)
|
1416.2(1.15)
|
∆Esurf
|
-13.4(0.02)
|
-14.5(0.02)
|
-13.9(0.02)
|
-13.5(0.02)
|
∆Ggas
|
-721.3(0.96)
|
-715.8(0.91)
|
-1058.5(1.09)
|
-1478.1(1.24)
|
∆Gsol
|
661.6(0.86)
|
653.0(0.86)
|
992.4(0.99)
|
1402.7(1.15)
|
∆Gbind (MMGBSA)
|
-59.7(0.28)
|
-62.8(0.23)
|
-66.1(0.21)
|
-75.4(0.23)
|
The effect of mutations can be investigated through the free energy calculations that track the influence of changes in certain positions 76. The results of the energy of decomposition by residue for RBDWT-ACE2, RBDAlpha-ACE2, RBDOmicron-ACE2 and RBDDelta-ACE2 complex demonstrate that the RBD is the region that has more energy variations, attractive and repulsive, when evaluated the electrostatic contributions (see Figure 4, Figure S4, Figure S5 and Figure S6). The evaluation of the decomposition energy per residue shows the mutations N440K, T478K, Q493R and Q498R observed in RBDOmicron provide favorable interaction between RBDOmicron and ACE2. Curiously, all these mutations include positively charged residues Lys or Arg (see Table 2). For example, K478 in RBDOmicron present a stabilization effect (-85.8 kcal/mol), while T478 in RBDWT has a destabilization effect (0.7 kcal/mol), see Table 2. Additionally, Table S2 shows the hydrogen bonds in the protein-protein interaction for the SARS-Cov-2, Alpha, Delta and Omicron system.
The N501Y mutation in the RBDAlpha has a very similar contribution to the native RBD system, indicating that this mutation does not cause such apparent changes in the energetic contributions, therefore the main feature that contributes to the better binding of RBDAlpha to ACE2, compared to the RBDWT, it is its conformational stability that differs from other spikes. The alterations in the Delta variant cause a highly attractive energy, in which the residue L352R had an energetic contribution of -90,524 kcal/mol and T478K equal to -82,654 kcal/mol (see table 2), indicating that there is a great improvement in the binding with the receptor. The mutations present in RBDOmicron demonstrate that during the gain in the energetic contribution of the residues.
Some mutations present in RBDOmicron (N440K, T478K, Q493R, Q498R) demonstrate that substitutions for positively charged residues guide an improvement in the contribution to the interaction with ACE2 (Figure S7). T478K is located in a more solvent-oriented region, allowing interaction with ACE2, due to the increase in the side chain Figure S7a. As well, the Q493R substitution allows favorable interaction with negatively charged residues of ACE2 such as Asp38 and Glu35, improving the binding with the receptor and increasing the affinity of the spike protein (Figure S7b). The N440K in the omicron is located in the region most focused on the solvent, increasing the contribution of this region with the medium (Figure S7c), whereas the Q498R substitution improves the protein-protein interaction since this contribution is 24 times greater in relation to the WT, demonstrating that these substitutions are essential for improving interaction with ACE2 (Figure S7d).
Table 2: Decomposition energies per residue in kcal/mol for the main mutation positions of RBD WT, Alpha, Delta and Omicron.
SARS-CoV-2
|
Alpha
|
|
Delta
|
|
Omicron
|
|
G339
|
0.7
|
|
0.8
|
|
0.8
|
G339D
|
68.4
|
S371
|
0.8
|
|
0.4
|
|
1.1
|
S371L
|
0.8
|
S373
|
1.0
|
|
0.6
|
|
0.9
|
S373P
|
0.6
|
S375
|
-0.3
|
|
-0.3
|
|
-0.1
|
S375F
|
-0.4
|
K417
|
-121.2
|
|
-131.5
|
|
-112.4
|
K417N
|
-2.3
|
N440
|
-0.4
|
|
-0.3
|
|
-0.2
|
N440K
|
-98.6
|
G446
|
0.3
|
|
-0.2
|
|
-0.2
|
G446S
|
1.2
|
L452
|
-0.7
|
|
-0.6
|
L452R
|
-90.5
|
L452
|
-1.4
|
S477
|
-1.1
|
|
-1.7
|
|
-1.4
|
S477N
|
-0.6
|
T478
|
0.7
|
|
-2.4
|
T478K
|
-82.6
|
T478K
|
-85.8
|
E484
|
88.2
|
|
94.4
|
|
93.8
|
E484A
|
0.1
|
Q493
|
-8.7
|
|
-11.6
|
|
-8.8
|
Q493R
|
-163.7
|
G496
|
-3.6
|
|
-3.1
|
|
-4.9
|
G496S
|
-6.3
|
Q498
|
-6.7
|
|
-2.1
|
|
-7.4
|
Q498R
|
-161.0
|
N501
|
-8.6
|
N501Y
|
-8.1
|
|
-10.1
|
N501Y
|
-2.2
|
Y505
|
-7.4
|
|
-5.5
|
|
-8.0
|
Y505H
|
-1.4
|