Molecular dynamic simulations unveil conformational dynamics of SARS-CoV-2 spike protein RBD and ACE2 receptor

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Researchers from the Federal University of Para, Brazil, presented a systematic analysis of the affinity and conformational dynamics between the binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) spike (S) protein and the host angiotensin-converting enzyme 2 (ACE2) receptor.

Study: Assessment of Mutations on RBD in the Spike Protein of SARS-CoV-2 Alpha, Delta and Omicron Variants. Image Credit: Akarat Jarernthanawut/Shutterstock

The researchers used accelerated molecular dynamics (aMD) simulations and free energy calculations to assess whether infectivity and transmission of SARS-CoV-2-related variants were related to binding to the receptor.

The study has been posted to the Research Square* preprint server and is currently under review at Scientific Reports journal.


SARS-CoV-2 S glycoprotein contains S1 and S2 subunits and is composed of a total of 1,273 residues.  The interaction between the RBD located in the S1 domain with ACE2 guides viral entry into the cell. S1 RBD underwent conformational changes in specific residues that revealed determinants of receptor binding.

Evidence from several studies suggested that mutations involving K417N, S477N, T478K, E484A, and N501Y in SARS-CoV-2 Omicron and other variants conferred viral escape capabilities or better binding affinity to ACE2 receptor and hence increased reinfection risk.

To analyze this further, the current study explored the effect of mutations in the S RBD of the SARS-CoV-2 Alpha, Delta, and Omicron variants on binding affinity to the human ACE2 receptor.

Study design

In this study, the authors performed classical molecular dynamics (cMD) simulations to assess average dihedral and total protein energies as a reference for aMD simulations. They carried out an aMD simulation of 200 ns to explore conformations over time for the RBDWT-ACE2, RBDAlpha-ACE2, RBDDelta-ACE2, and RBDOmicron-ACE2 complex systems.

The root-mean-square fluctuations (RMSF) analysis revealed the flexibility of each residue in the protein-protein complex system. Principal components analysis (PCA) allowed diagonalization of the covariance matrix to obtain the principal components. PCA graphs were built through combinations of PC1 vs PC2, PC2 vs PC3, and PC3 vs PC1 in which the clusters demonstrated two possible states for all systems in PC1 vs PC2.

The authors calculated binding free energy for RBD-ACE2 complexes of different SARS-CoV-2 variants to estimate SARS-CoV-2 affinity for human ACE2 receptor and potential risk of immune invasion by different SARS-CoV-2 variants. Decomposition energy assessed energy contribution of amino acid with the RBD and ACE2 binding, while the molecular mechanics, the generalized Born model, and solvent accessibility (MMGBSA) method implemented in AMBER calculated these free energies based on the greatest stability of the aMD trajectory.


The aMD simulations showed that RMSD of RBDWT-ACE2, RBDAlpha-ACE2, and RBDDelta-ACE2 complexes were in the 1 to 3 Å fluctuation range. However, the RBDOmicron-ACE2 complex represented different variations in the 1 to 4 Å range. Intriguingly, all the RBD-ACE2 complex systems had achieved structural equilibrium.

The RMSF analysis showed the greatest fluctuation of ACE2 in the regions of 123 to 178,  395 to 425, and 248 to 368 residues that move to interact with viral RBD. RBDalpha-ACE2 showed lower fluctuation as compared to the RBDWT, RBDDelta, and RBDOmicron.

The essential dynamic analysis demonstrated clusters in PCA graphs showing two probable states for all systems in PC1 vs PC2. However, the Alpha variant showed a higher cluster number wherein each time gap was differentiated into small clusters.

The initial structure of the RBDOmicron system differed from the final structure resulting in variations of the aMD structures. The RBDWT and the RBDOmicron strains elucidated substantial fluctuations in conformation; however, the RBDAlpha variant had greater stability.

The RBDOmicron showed the greatest binding affinity to the human ACE2 receptor and similar conformational fluctuation as compared to the other variants.  RBDOmicron showed a higher binding free energy (-75.4 kcal/mol) as compared to RBDDelta (-66.1 kcal/mol), RBDAlpha (62.7836 kcal/mol), and the RBDWT type (-59.7 kcal/mol).

Decomposition energy calculation showed that in RBDOmicron, mutations N440K, T478K, Q493R, and Q498R resulted in favorable interaction between RBDOmicron and ACE2.  Interestingly all mutations in RBDOmicron included positively charged residues of Lys or Arg. The Omicron K478 mutation (decomposition energy, -85.8 kcal/mol) had a stabilizing effect while T478 mutation in RBDWT (0.7 kcal/mol) had a destabilizing effect.

The T478K mutation was located in a solvent-oriented region and allowed ACE2 interaction due to the side chain increase. The Q493R substitution allowed a favorable interaction with Asp38 and Glu35 negatively charged residues of ACE2 increasing the S protein binding. N440K mutation located in the region focused on solvent whereas Q498R mutation improved protein-protein interaction and this contribution was 24 times higher as compared to RBDWT.

The N501Y mutation in RBDAlpha showed similar decomposition energy as compared to RBDWT. Hence, conformational stability in RBDAlpha was responsible for its better binding to ACE2. In RBDDelta, the L352R and T478K mutations had a higher energetic contribution of

-90.5 and -82.6 kcal/mol, respectively, highlighting increased binding improvement with the ACE2 receptor.


The findings of this study demonstrated that the SARS-CoV-2 RBDOmicron-ACE2 complex elucidated identical fluctuations as compared to the S protein from wild type, Alpha, and Delta strains.

Overall, mutations in the RBDOmicron increased S protein binding affinity for ACE2 and further mutations of uncharged residues to positively charged Lys and arg residues in a key position with RBD. This accounts for the high transmissibility of the SARS-CoV-2 Omicron variant compared to other variants.

*Important notice

Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Source: News Medical

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