Report reveals that the major clinical manifestations developed by the Omicron variant is "mild infection", including headache, body ache, muscles ache, cough, fever, generalized myalgia, and severe fatigue. Nevertheless, it is infecting younger and middle-aged groups [25]. More particular, the incidents of higher rate of infections are not associated with the severity of the infection outcome. At this time point, with the generation of new SARS CoV-2 variants like Delta followed by the Omicron need to be considered in association with some other factors like global vaccination, global pre-infection (by predecessor variants) and restrictions-lockdown. The interactive role of these factors may have some influences on the infection and disease severity by Omicron or other new variants. Report reveals that the sera collected from the Omicron infected person has a significant in vitro neutralization effect of Omicron variant with developing antibody response. Notwithstanding, there was an enhancement of several fold of Delta virus neutralization by this sera which may result in decreased ability of Deltato re-infect those individuals [26].
In the current study we are presenting by the in silico study the comparative analysis binding/docking parameters of SARS CoV-2 Wuhan, Delta and Omicron variants with human ACE2. We further analyzed the effects of the 59 amino acids of RBD fraction of the SARS CoV-2 and its several mutants THR500SER/GLY/ALA/CYS docking/blocking effects on ACE2 Omicron spike binding. Beside the individual spike variant binding with ACE2, comparative/combined binding of different spikes were also evaluated to predict which is more infective/transmissible. Our present results suggest few considerations.
1. SARS-COV2 and Omicron spike proteins binding with Human ACE2 receptor
The Spike glycoprotein’s of both variant initially binds to the Angiotensin Converting Enzyme 2 (ACE2) present at the host cell surface and then the viral entry gradually proceeds. This ACE2 attachments site remains folded until it reaches the receptor. Just before ACE2 attachment in SARS-CoV2 spike protein's flexible part become unfolded and the attachment site become exposed [27]. Individual docking of ACE2 and three different spike protein results were observed [figure 1]. According to the competitive multi docking studies between ACE2-SARSCoV2 – Omicron showed highest docking score of -194.5 +/- 5.1. The cumulative energy calculation of Van der Waals energy, desolvation energy and electrostatic energy represented the negative value of -98.0 +/- 9.9, -50.9 +/- 13.4 and -315.3 +/- 105.1 respectively [ Table S1a].
Electrostatic energy represents the potential energy of a system placed within the time-invariant electric field [28] where the positive value indicated the repulsion and negative value indicated the surface attraction between two molecules [29]. Here, SARS-CoV-2 showed higher binding affinity with ACE2 with a value -9 ΔG (kcal mol-1) and it also showed some Dissociation constant value of 2.50E-07. In contrary, molecular docking with ACE2 and Omicron showed comparable binding affinity of -7.4 ΔG (kcal mol-1) [Table S2a] at the binding site. The active site of ACE2 comprises of different amino acids, i.e. TYR83, GLN24, LYS31, GLU35, LYS353, ASP38 and LYS68 with which interaction of TYR489, AS487, TYR453, ARG403, ASP405, TYR505, GLY496 and ASN501 different amino acids of SARS-CoV2 binding sites were observed respectively [Figure 2; Table S3a].
The active site of ACE2 comprises of different amino acids, i.e. ASP67 and GLU75 with which interaction of LYS478, ASN487and TYR489 different amino acids of Omicron variant binding sites were observed respectively [Figure 2; Table S3a]. At the surface of ACE2 and spike glycoprotein interaction, all the amino acids interact with each other with the formation of H-bonds [Table S3a].
2. Delta and Omicron spike proteins binding with Human ACE2 receptor
Molecular docking with Delta variant and Omicron variant with ACE2 receptor revealed that the highest docking score value of -137.6 +/- 12.1. The calculation of Van der Waals energy, desolvation energy and electrostatic energy represented the negative value of -80.4 +/- 4.0, -31.8 +/- 2.9 and -284.8 +/- 64.1respectively [table S1a]. Here, DELTAvariant showed higher binding affinity with ACE2 with a value -9.7 ΔG (kcal mol-1) and it also showed some Dissociation constant value of 7.70E-08 [table S2a]. Contrarily, Omicron variant did not showany binding affinity against ACE2 in presence of the DELTA. It could be possible that the 15 mutations of RBDOmicron are not evenly distributed in RBD, but rather crowed in its RBM with 10 residues, viz., N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y and Y505H [30]. For that reason, Omicron variant have lower binding affinity than DELTA.
The active site of ACE2 comprises of different amino acids, i.e. GLN42, ASP38 and LYS353 with which interaction of ASP571, ARG567 and GLN563 different amino acids of Delta binding sites were observed respectively [Figure 3;Table S3a]. The H-bond length also observed between amino acid residues [Table S3a]. On the other hand, molecular docking with of ACE2 and Omicron showed no binding affinity and no binding occur [Table S2a].
3. Comparative study between 3 spike proteins binding with Human ACE2 receptor
According to the docking result between ACE2, Delta, Omicron and SARS-CoV-2 showed the highest docking score value of -250.8 +/- 19.4. The calculation of Van der Waals energy, desolvation energy and electrostatic energy represented the negative value of -157.9 +/- 11.4, -451.1 +/- 46.0 and -63.6 +/- 7.8 respectively [table S1a]. The binding affinity of DELTA, Omicron with ACE2 was -7.1 and -9.9 respectively [table S2a]. But molecular docking with ACE2 and SARS-CoV2 showed no binding affinity. From the above two results, we analyze that Delta is stronger binding affinity with ACE2 than Wild type SARS-CoV-2 and Omicron. When we study MD stimulation of ACE2 binding affinity with 3 different strains together, Omicron variant showed comparable binding affinity with human ACE2 in comparison with Delta strain.
According to Bloom et al [31], it was found that 9 RBDOmicron mutations (S371L, S373P, S375F, K417N, G446S, E484A, G496S, Q498R, Y505H) should decrease the binding affinity to ACE2 while the other 6 mutations (G339D, N440K, S477N, T478K, Q493K, N501Y) should increase the binding affinity, resulting in a challenge of predicting its transmissibility and potential immune evasion risk. The binding site of ACE2 comprises of different amino acids, i.e. GLU329, TYR41, TYR83 and SER19 with which interaction of LYS558, GLN563, LYS462 and GLU471 different amino acids of Delta binding sites were observed respectively [Figure 4; Table S3a]. The binding site of ACE2 comprises of different amino acids, i.e. GLU87 and LYS112 with which interaction of ARG498 and ALA372 different amino acids of Omicron binding sites were observed respectively [Figure 4; Table S3a]. The H-bond length also observed between amino acid residues [table S3a].
4. Measurement of binding affinity and kinetics of different CUTs segment
Here we investigate the blind docking of Wild 59, T500S, T500C, T500G and T500A interaction with ACE2 and Omicron showed the docking score value of -175.1 +/- 13.8,-163.1 +/- 13.8, -172.2 +/- 26.8 ,-163.6 +/- 9.4 and -174.0 +/- 11.9 respectively [table S1b].According to Binding affinity result, the binding affinity between Wild 59 segments and Omicron variant was -12.5 ΔG (kcal mol-1), T500S and Omicron variant -16.4 ΔG (kcal mol-1), T500C and Omicron variant -12.2 ΔG (kcal mol-1),T500G and Omicron variant -13 ΔG (kcal mol-1) and T500A and Omicron variant -12.3 ΔG (kcal mol-1) [table S2b].
Among the mutated CUTs segment, we showed that the best binding affinity between CUTs and spike protein was -16.4 ΔG (kcal mol-1) because the lower energy and higher negative value means the higher stability of the complex. Due to the sequence shorting may be T500S cut have higher binding affinity than 84 cut segments. So, we choose only T500S cut segment for further analysis and Wild 59 cut segment as a control. After analysis the H- bond pattern, T500S cut showed 6 interactions with Omicron spike protein at RBD site ranged from 1.9 -2.1Å [table S3b], whereas Wild 59 cut showed 5 interactions ranged from 1.9- 2.8Å [table S3b]. The binding affinity values and H-bond interactions of T500S were comparatively higher than the Wild 59-cut.
The interaction site of T500S cut comprises of different amino acids ASN137, TYR 185, VAL 187, ASN 164 and SER 154with which interaction of ASN450, TYR501, PRO499, CYS488, GLY485 and ALA484 different amino acids of Omicron binding sites were observed respectively[Figure 6; Table S3b]. Our previous study analyze that the CUT segments of 84 amino acids after molecular docking with ACE2 receptor and SARS-CoV-2, the docked structures were either partial or complete interactive distortions of spike glycoprotein from ACE2 receptor was observed. But when we observed the docking structure of T500S cut segment, Omicron and ACE2, T500S cut bind at RBD site of Omicron and ACE2 bind different position of Omicron not in the RBD active site. The docking structure of WILD59 segment Omicron and ACE2 was shown WILD59 segment bind at RBD site instead of ACE2 receptor [Figure 5; Table S3b].
5. Molecular dynamics simulation
The result of molecular dynamics simulation and normal mode analysis (NMA) of different docked complex of ACE2 and different variants of SARS-CoV 2 is illustrated in Fig 7. The simulation study was conducted to determine the movements of protein molecules. The main-chain deformability graph of the three complexes is shown in S-Fig:1, illustrates the peaks in which represents the regions of the protein with deformability. The locations with hinge regions are high deformability. The B-factor is a measure for flexibility in a protein and quantifies the uncertainty of each. The B-factor values calculated by normal mode analysis are proportional to root mean square shown in Fig 7. B-factor values quantify the uncertainty of each atom. The B-factor graph gives a clear visualization of the relation of the docked complex between the NMA and the PDB sector. The covariance matrix between the pairs of residues is shown in S-Fig:1, indicating their correlations (correlated motion indicated by red color; uncorrelated motion indicating by white color; anti correlated motion indicated by blue color).
We also examined the molecular dynamics simulation of wild59 CUTs and T500S 59 CUTs with Omicron variant is illustrated in Figure 8. The B-factor is a measure for flexibility in a protein and quantifies the uncertainty of each. The B-factor values calculated by normal mode analysis are proportional to root mean square shown in Figure 8. B-factor values quantify the uncertainty of each atom. The B-factor graph gives a clear visualization of the relation of the docked complex between the NMA and the PDB sector. The main-chain deformability graph of the three complexes is shown in S-Fig: 2, illustrates the peaks in which represents the regions of the protein with deformability. The locations with hinge regions are high deformability. The covariance matrix between the pairs of residues is shown in S-Fig: 2, indicating their correlations (correlated motion indicated by red color; uncorrelated motion indicating by white color; anti correlated motion indicated by blue color). So, our all docking complexes are in stable form.
SARS-CoV-2 Omicron RBD shows weaker binding affinity than the currently dominant Delta variant to human ACE2 [32]. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related corona viruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates [33].
Report reveals that infection to Omicron caused an enhancement of Delta virus neutralization, which increased 4.4-fold. This may result in decreased ability of Delta to re-infect those individuals [34]. Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles [35]. Mutations on RBD of SARS-CoV-2 Omicron variant result in stronger binding to human ACE2 receptor [36].
In conclusion, the present 59 amino-acid cut RBD fraction was found to be good blocker of the spike-ACE2 binding. Moreover, introduced mutations especially T500S and T500G were highly potent with very high binding affinity to block the ACE2 and Omicron spike binding. The present investigation may explain Delta variant is significantly stronger than WT and Omicron, while there is no significant difference between Wild type and Omicron but more research is required to explore its less ability to penetrate the lung tissue. This work may not explain the replication/propagation or immuno-invasion ability of omicron. In this case, lung TMPRSS2 may have some role in viral internalization. In regard to the last two years’ strategies, global vaccination and natural infection, the disease outcome and severities of different new variants become less predictable. Further studies are required in this regard.