Assessment of Mutations on RBD in the Spike Protein of SARS-CoV-2 Alpha, Delta and Omicron Variants

Assessment of Mutations on RBD in the Spike Protein of SARS-CoV-2 Alpha, Delta and Omicron Variants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of Mutations on RBD in the Spike Protein of SARS-CoV-2 Alpha, Delta and Omicron Variants Clauber Henrique Souza Costa, Camila Auad Beltrão Freitas, Cláudio Nahum Alves, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1401835/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) variant Omicron spread more rapid than the other variants of SARS-CoV-2 virus. Mutations on the spike (S) protein receptor-binding domain (RBD) are critical for the antibody resistance and infectivity of the SARS-CoV-2 variants. In this study, we have used accelerated molecular dynamics (aMD) simulations and free energy calculations to present a systematic analysis of the affinity and conformational dynamics along with the interactions that drive the binding between Spike protein RBD and ACE2 receptor. We evaluate the impacts of the key mutation that occur in the RBDs Omicron and other variants in the binding with the human ACE2 receptor. The results shows that S protein Omicron have stronger binding to the ACE2 than other variants. The evaluation of the decomposition energy per residue shows the mutations N440K, T478K, Q493R and Q498R observed in Spike protein of SARS-CoV-2 provided a stabilization effect for the interaction between the SARS-CoV-2 RBD and ACE2. Overall, the results demonstrate that faster spreading of SARS-CoV-2 omicron may be correlated with binding affinity of S protein RBD to ACE2 and mutations of uncharged residues to positively charged residues such as Lys and Arg in key positions in the RBD. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction First reported in the city ​​of Wuhan, China 1,2 , Coronavirus disease (COVID-19) named by World Health Organization (WHO) was declared a global pandemic on March 2020 3 . COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 4,5 1,2 The spread of SARS-CoV-2 have cost millions of lives and caused many implications for health, society and the economy 6,7 . In January 2022, the WHO reported over 304 million confirmed cases of COVID-19 and over 5.4 million fatalities have been reported since the beginning of the outbreak 8 . Vaccines are effective for reducing the number deaths by COVID-19 9–11 . On the other hand, variants may cause impact on the virus recognition by antibody-mediated vaccines 12–14 . Different mutations have been reported in the gene encoding the S protein of SARS-CoV-2 15,16 , and recently, the world have faced rapid increase in COVID-19 mediated by new variants 17 . The last variant detected was named Omicron (B.1.1.529) 18 , identified in numerous countries in November 2021, first reported in South African with a large number of mutations, including K417N, S477N, T478K, E484A, and N501Y, which are also found in other variants 19–21 and evidences suggests there may be an increased risk of reinfection involving this variant 22,23 due to improve viral escape or binding affinity to angiotensin-converting enzyme 2 (ACE2) 24–26 . A recent study reported that 45 point mutations was identified and found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants 27 . Additionally, These mutations and deletions in the S-protein sequence can alter the structure, affecting its stability and function, further exacerbating SARS-CoV-2 infectivity 16,28 . However, N501Y mutation is a key contact residue in the RBD, enhancing virus binding affinity to ACE2 29–31 making the virus more contagious and the deletions H69/V70 is required for increase optimal infectivity of Alpha variant, that drives by higher levels of spike incorporation into virions 32 . Coronaviruses use spike glycoprotein, with S1 subunit and S2 subunit in each spike monomer, anchored in the virion envelope to bind to their cellular receptors 33,34 and mediates the recognition of the host-cell receptors and facilitates the cell attachment (S1 subunit) and the cell membrane fusion (S2 subunit) during the viral infection 35 . The receptor-binding domain (RBD) located in the S1 region (318 to 510 sequence region) performs strongly binds to the peptidase domain of ACE2 36,37 , leading to a critical virus-receptor interaction and reflects viral host range, tropism and infectivity 38 . the RBD of S1 undergoes conformational changes that transiently conceal or reveal the determinants of receptor binding 24,39 The spike (S) protein of SARS-CoV-2 consists in an extracellular N-terminus, a transmembrane (TM) domain and a intracellular C-terminal segment 40 . S protein has a total length of 1273 amino acids 35 and molecular weight of 180–200 kDa 41 . It has a signal peptide (1–13) at the N-terminus, followed by S1 subunit (14–685) and the S2 subunit (686–1273) 42 . The structure of the RBD allows for ways to alter its genetic material, developing variants by the changes in spike protein amino acids and as viruses replicate 16 , copying errors of itself, resulting in mutations that arise in their genomes generating several strains of SARS-CoV-2 17,43 that differ in transmission, infectivity and severity of the disease 44 . ACE2 primary physiological role is in the maturation of angiotensin (Ang) 45 , a peptide hormone that controls vasoconstriction and blood pressure, is a type I membrane protein expressed in lungs, heart, kidneys, and intestine 25,46 , thereat, decreased expression of ACE2 is associated with cardiovascular diseases 47 . The structural features of RBD increase its binding affinity to the ACE2 receptor and it is a significant step for SARS-CoV-2 to enter into target cells 33,48 . Computer modelling studies of the interaction between the SARS-CoV-2 RBD and ACE2 were able to identify the residues involved in this interaction and elucidate how the structural change benefits receptor recognition and virus entry into the host cell, that occurs by proteolytic processing of the spike protein to promote cell-virus fusion 49 . Therefore, atomic details may clarify the importance and significance of investigating the changes in residues that facilitate efficient cross-species infection and human-to-human transmission 34 . Whereas the essential evolution and consequent mutation of SARS-CoV-2 takes place remotely from the RBD in the spike protein, such evolution may facilitate the conformational change in specific residues, punctually interfering with the infection process that occurs after the virus binds to ACE2 50 . Recently, Warshel and co-workers studied the mechanism of the binding affinity changes for mutations at different spike protein domains of SARS-CoV-2 using coarse-grained potential surface 51 . More recently, Chen and co-workers used machine learning model to analyze how the RBD mutations on the Omicron variant may affect the viral infectivity and efficacy of existing vaccines and antibody drugs 52 . In this study, we have used all-atom accelerated molecular dynamics simulations 53,54 to explore the impacts of the substitutions that occur in the Spike RBD of Alpha, Delta and Omicron variants in the binding with the human ACE2 receptor. In order to address the question whether variant infectivity and spreading is related to its binding to the receptor. Methods SARS-CoV-2 Spike protein (S protein) is a class I fusion homotrimer glycoprotein that is composed a total length 1273 residues 55 and the binding between the virus and the host cell is mediated by the interaction of the protein S receptor binding domain (RBD, located in the S1 domain) with the angiotensin converting enzyme receptor 2 (ACE2). Here, for the sake of simplicity, S protein RBD from SARS-CoV-2 was renamed as RBD x , where x represents the identification of each SARS-CoV-2 variant. The initial systems were built considering the coordinates of the RBD complex and the ACE2 (PDB code 6M0J) 33 . The protonation states of the protein residues were defined through the propKa program at pH 7 56 . The amino acids were treated with the ff14SB force field 57 using TLeap module included in AMBER 16 58 . Each system was solvated using TIP3P water 59 model in a cubic box with 10.0 Å of the amino acid at the end for all Cartesian directions. Then, each system was neutralized using Na + as contra-ions. We used four minimization steps with 10.000 cycles for each step, applying minimization first to water, contra-ions and protein, in the last step the minimization was applied to all atoms in the system in order to decrease energy, adjust interactions and decrease contacts with conjugate gradient and steepest descent. The systems were heated linearly from 0 to 300 K (tempi = 0.0; temp0 = 300.0) to avoid excessive and sudden fluctuations of the solute in a time of 5ns in NVT essemble employing Langevin dynamics as thermostat (collision frequency of 2 ps) had been used to guarantee a system equilibrium. The SHAKE algorithm 60 was employed to constraints all bonds involving hydrogen atoms. First, we have performed 10 ns of Classical molecular dynamics (cMD) simulations to calculate the average dihedral and total potentials energies to be taken as reference for the accelerated molecular dynamics (aMD) simulations. Then 200 ns of aMD simulations was carried out for each system: RBD WT -ACE2, RBD alpha -ACE2, RBD Omicron -ACE2 and RBD Delta -ACE2 complex in NPT essemble. The aMD technique is used to enhance sampling in the protein's conformational space, artificially reducing the energy barriers that separate different states of a given system 53,54,61–64 . Additionally, we used the Bio3D package 65 to perform the principal component analysis (PCA). The PCs were obtained from the diagonalization of the covariance matrix obtained from the Cartesian coordinates of the superposed Cα atoms of complex structure. To avoid an underestimate of the atomic displacement, an iterated superposition procedure was applied before the PCA, where residues displaying the largest positional differences were excluded at each round until only the invariant ‘core’ residues remained 66–69 . Protein -protein binding free energy In this study, we also evaluated the binding energy differences between the complexes and then the decomposition energy was added to assess the energy contribution of each amino acid during the binding of RBD to ACE2. The binding free energy for the each RBD-ACE2 complex was obtained using: ∆G bind = G RBD−ACE2 - G RBD – G ACE2 (1) Here, G RBD−ACE2 represent the average over the snapshots of a single trajectory of the MD RBD-ACE2complex, G RBD and G ACE2 corresponds to the free energy of RBD and ACE2 protein, respectively. The binding free energy was obtained using MMGBSA method 70,71 implemented in AMBER 16 58 . In order to calculate free energy with MMGBSA (Eq. 2 ) 5000 frames were taken from the 10 ns of MD production using: 72–74 is important to note that the entropy contribution was not included in the calculations due to the difficulty of accurately calculating entropy for a large protein-protein complex. 75 It is also worth to note that the frames were taken from the most stable structure observed in PCA graphics. Results And Discussion 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: RBD WT -ACE2, RBD alpha -ACE2, RBD Omicron -ACE2 and RBD Delta -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. RBD WT -ACE2, RBD alpha -ACE2 and and RBD Delta -ACE2 complexes were within fluctuation in a range of 1 to 3 Å (Figure 1), while the RBD Omicron -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 RBD alpha 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 RBD WT and the RBD omicron variant present greater conformational fluctuations, however, the RBD alpha variant stands out for its greater stability. In PC1 there are not many movements in RBD and ACE2 (Figure 3). The main movement of RBD WT and RBD omicron 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 (∆G bind (MMGBSA)) based on the points of greatest stability of the aMD trajectory (see Table 1). The RBD omicron shows the highest binding affinity to ACE2, reflecting the infectivity process, but its conformational fluctuations is similar to the other variants. RBD omicron present an adaptive and non-aggressive process when compared to the RBD alpha (with free energy of binding equal to -62.7836 kcal/mol), which demonstrated the lower free energy than RBD WT (-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 RBD Delta has a higher binding affinity with the human receptor compared to the RBD WT (-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 ∆G bind in comparison to RBD WT . 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 ∆E vdw -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) ∆E GB 675.0(0.87) 667.5(0.87) 1006.3(1.01) 1416.2(1.15) ∆E surf -13.4(0.02) -14.5(0.02) -13.9(0.02) -13.5(0.02) ∆G gas -721.3(0.96) -715.8(0.91) -1058.5(1.09) -1478.1(1.24) ∆G sol 661.6(0.86) 653.0(0.86) 992.4(0.99) 1402.7(1.15) ∆G bind (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 RBD WT -ACE2, RBD Alpha -ACE2, RBD Omicron -ACE2 and RBD Delta -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 RBD Omicron provide favorable interaction between RBD Omicron and ACE2. Curiously, all these mutations include positively charged residues Lys or Arg (see Table 2). For example, K478 in RBD Omicron present a stabilization effect (-85.8 kcal/mol), while T478 in RBD WT 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 RBD Alpha 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 RBD Alpha to ACE2, compared to the RBD WT , 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 RBD Omicron 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 Conclusion In this study, we evaluated the effect of residues mutation on structural and energetics of Spike protein RBD from SARS-CoV-2 variants in complex with the human ACE2 receptor. All-atoms accelerated Molecular Dynamics simulations and PCA analysis shows that that the RBD Omicron -ACE2 complex present similar fluctuation in comparison to S protein from Native, Delta and Alpha variants. The binding affinity of each RBD x to ACE2 was obtained using MM-GBSA methods. The results shows that the trend in the calculated binding free energies correlates well with virus infectivity of each variant. The mutation in RBD Omicron increase the affinity of Spike protein for ACE2 and may explain Omicron's high transmissibility in comparison with other SARS-CoV-2 variants. The stabilization effect RBD Omicron -ACE2 complex is achieved manly due the substitution of uncharged residues by positively charged residues: Lys and Arg in key positions. Overall, our results may explain at molecular level the effect of key mutations in the Spike protein for virus infectivity. Declarations Data availability All necessary files to conduct this work (.pdb and .parm7) can be found attached as the Supporting Information. The AMBER18 suite of programs and the Amber ff14SB force field were used to carry out the MD simulations and can found at https://ambermd.org/. Acknowledgements Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their financial support. We also thank the access of the computational resources of the Supercomputer Santos Dumont (SDumont) provided by the Laboratório de Computação Científica (LNCC), Apollo 2000 and CABANO – UFPA. Author information These authors contributed equally: Clauber Henrique Souza da Costa, Camila Auad Beltrão de Freitas. References Wang, D. et al. Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus–Infected Pneumonia in Wuhan, China. JAMA 323 , 1061–1069 (2020). Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395 , (2020). Who, W. H. O. Coronavirus disease (COVID-19) outbreak (World Health Organization, 2020). 2020 (2020). Wu, F. et al. A new coronavirus associated with human respiratory disease in China. 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Omicron Variant ( B . 1 . 1 . 529 ): Infectivity , Vaccine Breakthrough , and Antibody Resistance. (2021). doi:10.1021/acs.jcim.1c01451 Hamelberg, D., Mongan, J. & McCammon, J. A. Accelerated molecular dynamics: a promising and efficient simulation method for biomolecules. J. Chem. Phys. 120 , 11919–11929 (2004). Kukol, A. Molecular modeling of proteins: Second edition . Molecular Modeling of Proteins: Second Edition 1215 , (2014). Watanabe, Y., Allen, J. D., Wrapp, D., McLellan, J. S. & Crispin, M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science 369 , 330–333 (2020). Søndergaard, C. R., Olsson, M. H. M., Rostkowski, M. & Jensen, J. H. Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of pKa Values. J. Chem. Theory Comput. 7 , 2284–2295 (2011). Maier, J. A. et al. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 11 , 3696–3713 (2015). D.A. Case, R.M. Betz, D.S. 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Computational study of conformational changes in human 3-hydroxy-3-methylglutaryl coenzyme reductase induced by substrate binding. J. Biomol. Struct. Dyn. 37 , 4374–4383 (2019). Costa, C. H. S. da et al. Unraveling the conformational dynamics of glycerol 3-phosphate dehydrogenase, a nicotinamide adenine dinucleotide-dependent enzyme of Leishmania mexicana. J. Biomol. Struct. Dyn. 1–12 (2020). doi:10.1080/07391102.2020.1742206 Grosso, M., Kalstein, A., Parisi, G., Roitberg, A. E. & Fernandez-Alberti, S. On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein. J. Chem. Phys. 142 , 245101 (2015). Srinivasan, J., Cheatham, T. E., Cieplak, P., Kollman, P. A. & Case, D. A. Continuum Solvent Studies of the Stability of DNA, RNA, and Phosphoramidate−DNA Helices. J. Am. Chem. Soc. 120 , 9401–9409 (1998). Kollman, P. a et al. Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc. Chem. Res. 33 , 889–97 (2000). Lill, M. A. & Thompson, J. J. Solvent interaction energy calculations on molecular dynamics trajectories: increasing the efficiency using systematic frame selection. J. Chem. Inf. Model. 51 , 2680–2689 (2011). Case, D. A. et al. The Amber biomolecular simulation programs. J. Comput. Chem. 26 , 1668–1688 (2005). Cui, Q. et al. Molecular Dynamics—Solvated Interaction Energy Studies of Protein–Protein Interactions: The MP1–p14 Scaffolding Complex. J. Mol. Biol. 379 , 787–802 (2008). Yang, Y., Liu, H. & Yao, X. Understanding the molecular basis of MK2-p38α signaling complex assembly: insights into protein-protein interaction by molecular dynamics and free energy studies. Mol. Biosyst. 8 , 2106–2118 (2012). Bai, C. et al. Predicting Mutational Effects on Receptor Binding of the Spike Protein of SARS-CoV-2 Variants. J. Am. Chem. Soc. 143 , 17646–17654 (2021). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1401835","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":91201251,"identity":"b846d2cd-27c0-4c36-8aae-72be209914b5","order_by":0,"name":"Clauber Henrique Souza Costa","email":"","orcid":"","institution":"Federal University of Para","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Clauber","middleName":"Henrique Souza","lastName":"Costa","suffix":""},{"id":91201253,"identity":"bc45d523-66c2-44d3-88eb-4505bfbd5774","order_by":1,"name":"Camila Auad Beltrão Freitas","email":"","orcid":"","institution":"Federal University of Para","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Camila","middleName":"Auad Beltrão","lastName":"Freitas","suffix":""},{"id":91201254,"identity":"c5ac0fb3-8670-482c-8cdf-3066e5e7f58d","order_by":2,"name":"Cláudio Nahum Alves","email":"","orcid":"","institution":"Federal University of Para","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cláudio","middleName":"Nahum","lastName":"Alves","suffix":""},{"id":91201256,"identity":"4004d43a-980b-42a6-812b-f5aeb83e0505","order_by":3,"name":"Jerônimo Lameira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACPhCRwMAgx8B8gIGBsYEILWxQLcYMbAmkaAGCxAbitbCfTnzwcI9d+oZjvAc/MO64R4QWntzNBgnPknM3HONLlmA8U0yMw3K3SSQcYM7dcL/HQIKxLYEILfxvt/9IOFCfbnCMx/gHcVokcrcxJBw4nADUYkakLRJvNwMddtxw5jG+NIvEM0Ro4efP3fjxx4Fqeb5jvIdvfNxBhBYkwAOOU1K1jIJRMApGwSjABgAdajmfydmc9QAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Para","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jerônimo","middleName":"","lastName":"Lameira","suffix":""}],"badges":[],"createdAt":"2022-02-27 22:29:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1401835/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1401835/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19344008,"identity":"b2ab8de2-02cc-4c94-872d-d907b0cce9d5","added_by":"auto","created_at":"2022-03-17 18:36:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114360,"visible":true,"origin":"","legend":"\u003cp\u003eRMSD for RBD\u003csub\u003eWT\u003c/sub\u003e-ACE2, RBD\u003csub\u003ealpha\u003c/sub\u003e-ACE2, RBD\u003csub\u003eOmicron\u003c/sub\u003e-ACE2 and RBD\u003csub\u003eDelta\u003c/sub\u003e-ACE2 complexes.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1401835/v1/30ccbe0f44e6a33e5d7268f4.png"},{"id":19344009,"identity":"2f4db301-831d-4162-8946-7d09bcde3dc4","added_by":"auto","created_at":"2022-03-17 18:36:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":796290,"visible":true,"origin":"","legend":"\u003cp\u003ethree-dimensional structure of ACE2 and RBD with RMSF regions for SARS-CoV-2, Alpha, Delta and Omicron systems.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1401835/v1/0ce646b08cae8c03668d9d98.png"},{"id":19344010,"identity":"3fe8b2ba-516f-477a-9cde-791d87709acc","added_by":"auto","created_at":"2022-03-17 18:36:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6801990,"visible":true,"origin":"","legend":"\u003cp\u003eMovements described for the first principal component (PC1) for each structure of ACE2 and RBD. A) moving in PC1 to the native RBD complex (SARS-CoV-2) and ACE2 receptor. B) changes in PC1 to RBD\u003csub\u003eAlpha\u003c/sub\u003e and ACE2. C) Change moving of PC1 to RBD\u003csub\u003eDelta\u003c/sub\u003e and ACE2. D) moving from PC1 to the complex between RBD\u003csub\u003eOmicron\u003c/sub\u003e and ACE2. In turquoise, the initial structure of the movement, in dark magenta, the final structure and in gray, the intermediate structures of the movement. The conformational dynamics were obtained from 200 ns of aMD simulations.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1401835/v1/8f561d7cda3d483c21387794.png"},{"id":19344527,"identity":"02a2c4df-b994-4241-8e19-49b794c044e4","added_by":"auto","created_at":"2022-03-17 18:39:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2496106,"visible":true,"origin":"","legend":"\u003cp\u003ea) three-dimensional structure of the RBD\u003csub\u003eWT\u003c/sub\u003e and ACE2 complex with the electrostatic energy regions. B) Decomposition energy per residue for the RBD\u003csub\u003eWT\u003c/sub\u003e system connected to ACE2. The label in orange is from the ACE2 region and in purple is from RBD.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1401835/v1/a4eb41d80385eff88d561950.png"},{"id":19344528,"identity":"2fedaefb-857a-422d-8178-3c4aa6a09669","added_by":"auto","created_at":"2022-03-17 18:39:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":387884,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1401835/v1/6085f6c6-3b87-4bd9-9b52-b7642701bbf8.pdf"},{"id":19344012,"identity":"e32d2955-09b4-4316-a7b0-de9bd7f7798f","added_by":"auto","created_at":"2022-03-17 18:36:37","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4297605,"visible":true,"origin":"","legend":"","description":"","filename":"SISARS100322.docx","url":"https://assets-eu.researchsquare.com/files/rs-1401835/v1/fd0556e008304c80aca7ea98.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of Mutations on RBD in the Spike Protein of SARS-CoV-2 Alpha, Delta and Omicron Variants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFirst reported in the city ​​of Wuhan, China \u003csup\u003e1,2\u003c/sup\u003e, \u003cem\u003eCoronavirus disease\u003c/em\u003e (COVID-19) named by World Health Organization (WHO) was declared a global pandemic on March 2020 \u003csup\u003e3\u003c/sup\u003e. COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). \u003csup\u003e4,5 1,2\u003c/sup\u003e The spread of SARS-CoV-2 have cost millions of lives and caused many implications for health, society and the economy \u003csup\u003e6,7\u003c/sup\u003e. In January 2022, the WHO reported over 304\u0026nbsp;million confirmed cases of COVID-19 and over 5.4\u0026nbsp;million fatalities have been reported since the beginning of the outbreak \u003csup\u003e8\u003c/sup\u003e. Vaccines are effective for reducing the number deaths by COVID-19 \u003csup\u003e9\u0026ndash;11\u003c/sup\u003e. On the other hand, variants may cause impact on the virus recognition by antibody-mediated vaccines \u003csup\u003e12\u0026ndash;14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDifferent mutations have been reported in the gene encoding the S protein of SARS-CoV-2 \u003csup\u003e15,16\u003c/sup\u003e, and recently, the world have faced rapid increase in COVID-19 mediated by new variants \u003csup\u003e17\u003c/sup\u003e. The last variant detected was named Omicron (B.1.1.529) \u003csup\u003e18\u003c/sup\u003e, identified in numerous countries in November 2021, first reported in South African with a large number of mutations, including K417N, S477N, T478K, E484A, and N501Y, which are also found in other variants \u003csup\u003e19\u0026ndash;21\u003c/sup\u003e and evidences suggests there may be an increased risk of reinfection involving this variant \u003csup\u003e22,23\u003c/sup\u003e due to improve viral escape or binding affinity to angiotensin-converting enzyme 2 (ACE2) \u003csup\u003e24\u0026ndash;26\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA recent study reported that 45 point mutations was identified and found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants \u003csup\u003e27\u003c/sup\u003e. Additionally, These mutations and deletions in the S-protein sequence can alter the structure, affecting its stability and function, further exacerbating SARS-CoV-2 infectivity \u003csup\u003e16,28\u003c/sup\u003e. However, N501Y mutation is a key contact residue in the RBD, enhancing virus binding affinity to ACE2 \u003csup\u003e29\u0026ndash;31\u003c/sup\u003e making the virus more contagious and the deletions H69/V70 is required for increase optimal infectivity of Alpha variant, that drives by higher levels of spike incorporation into virions \u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCoronaviruses use spike glycoprotein, with S1 subunit and S2 subunit in each spike monomer, anchored in the virion envelope to bind to their cellular receptors \u003csup\u003e33,34\u003c/sup\u003e and mediates the recognition of the host-cell receptors and facilitates the cell attachment (S1 subunit) and the cell membrane fusion (S2 subunit) during the viral infection \u003csup\u003e35\u003c/sup\u003e. The receptor-binding domain (RBD) located in the S1 region (318 to 510 sequence region) performs strongly binds to the peptidase domain of ACE2 \u003csup\u003e36,37\u003c/sup\u003e, leading to a critical virus-receptor interaction and reflects viral host range, tropism and infectivity \u003csup\u003e38\u003c/sup\u003e. the RBD of S1 undergoes conformational changes that transiently conceal or reveal the determinants of receptor binding \u003csup\u003e24,39\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe spike (S) protein of SARS-CoV-2 consists in an extracellular N-terminus, a transmembrane (TM) domain and a intracellular C-terminal segment \u003csup\u003e40\u003c/sup\u003e. S protein has a total length of 1273 amino acids\u003csup\u003e35\u003c/sup\u003e and molecular weight of 180\u0026ndash;200 kDa \u003csup\u003e41\u003c/sup\u003e. It has a signal peptide (1\u0026ndash;13) at the N-terminus, followed by S1 subunit (14\u0026ndash;685) and the S2 subunit (686\u0026ndash;1273)\u003csup\u003e42\u003c/sup\u003e. The structure of the RBD allows for ways to alter its genetic material, developing variants by the changes in spike protein amino acids and as viruses replicate\u003csup\u003e16\u003c/sup\u003e, copying errors of itself, resulting in mutations that arise in their genomes generating several strains of SARS-CoV-2 \u003csup\u003e17,43\u003c/sup\u003e that differ in transmission, infectivity and severity of the disease\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eACE2 primary physiological role is in the maturation of angiotensin (Ang)\u003csup\u003e45\u003c/sup\u003e, a peptide hormone that controls vasoconstriction and blood pressure, is a type I membrane protein expressed in lungs, heart, kidneys, and intestine \u003csup\u003e25,46\u003c/sup\u003e, thereat, decreased expression of ACE2 is associated with cardiovascular diseases \u003csup\u003e47\u003c/sup\u003e. The structural features of RBD increase its binding affinity to the ACE2 receptor and it is a significant step for SARS-CoV-2 to enter into target cells \u003csup\u003e33,48\u003c/sup\u003e. Computer modelling studies of the interaction between the SARS-CoV-2 RBD and ACE2 were able to identify the residues involved in this interaction and elucidate how the structural change benefits receptor recognition and virus entry into the host cell, that occurs by proteolytic processing of the spike protein to promote cell-virus fusion \u003csup\u003e49\u003c/sup\u003e. Therefore, atomic details may clarify the importance and significance of investigating the changes in residues that facilitate efficient cross-species infection and human-to-human transmission \u003csup\u003e34\u003c/sup\u003e. Whereas the essential evolution and consequent mutation of SARS-CoV-2 takes place remotely from the RBD in the spike protein, such evolution may facilitate the conformational change in specific residues, punctually interfering with the infection process that occurs after the virus binds to ACE2 \u003csup\u003e50\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, Warshel and co-workers studied the mechanism of the binding affinity changes for mutations at different spike protein domains of SARS-CoV-2 using coarse-grained potential surface \u003csup\u003e51\u003c/sup\u003e. More recently, Chen and co-workers used machine learning model to analyze how the RBD mutations on the Omicron variant may affect the viral infectivity and efficacy of existing vaccines and antibody drugs\u003csup\u003e52\u003c/sup\u003e. In this study, we have used all-atom accelerated molecular dynamics simulations \u003csup\u003e53,54\u003c/sup\u003e to explore the impacts of the substitutions that occur in the Spike RBD of Alpha, Delta and Omicron variants in the binding with the human ACE2 receptor. In order to address the question whether variant infectivity and spreading is related to its binding to the receptor.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eSARS-CoV-2 Spike protein (S protein) is a class I fusion homotrimer glycoprotein that is composed a total length 1273 residues \u003csup\u003e55\u003c/sup\u003e and the binding between the virus and the host cell is mediated by the interaction of the protein S receptor binding domain (RBD, located in the S1 domain) with the angiotensin converting enzyme receptor 2 (ACE2). Here, for the sake of simplicity, S protein RBD from SARS-CoV-2 was renamed as RBD\u003csub\u003ex\u003c/sub\u003e, where x represents the identification of each SARS-CoV-2 variant. The initial systems were built considering the coordinates of the RBD complex and the ACE2 (PDB code 6M0J) \u003csup\u003e33\u003c/sup\u003e. The protonation states of the protein residues were defined through the propKa program at pH 7 \u003csup\u003e56\u003c/sup\u003e. The amino acids were treated with the ff14SB force field \u003csup\u003e57\u003c/sup\u003e using TLeap module included in AMBER 16 \u003csup\u003e58\u003c/sup\u003e. Each system was solvated using TIP3P water \u003csup\u003e59\u003c/sup\u003e model in a cubic box with 10.0 \u0026Aring; of the amino acid at the end for all Cartesian directions. Then, each system was neutralized using Na\u003csup\u003e+\u003c/sup\u003e as contra-ions.\u003c/p\u003e\n\u003cp\u003eWe used four minimization steps with 10.000 cycles for each step, applying minimization first to water, contra-ions and protein, in the last step the minimization was applied to all atoms in the system in order to decrease energy, adjust interactions and decrease contacts with conjugate gradient and steepest descent. The systems were heated linearly from 0 to 300 K (tempi\u0026thinsp;=\u0026thinsp;0.0; temp0\u0026thinsp;=\u0026thinsp;300.0) to avoid excessive and sudden fluctuations of the solute in a time of 5ns in NVT essemble employing Langevin dynamics as thermostat (collision frequency of 2 ps) had been used to guarantee a system equilibrium. The SHAKE algorithm \u003csup\u003e60\u003c/sup\u003e was employed to constraints all bonds involving hydrogen atoms.\u003c/p\u003e\n\u003cp\u003eFirst, we have performed 10 ns of Classical molecular dynamics (cMD) simulations to calculate the average dihedral and total potentials energies to be taken as reference for the accelerated molecular dynamics (aMD) simulations. Then 200 ns of aMD simulations was carried out for each system: RBD\u003csub\u003eWT\u003c/sub\u003e-ACE2, RBD\u003csub\u003ealpha\u003c/sub\u003e-ACE2, RBD\u003csub\u003eOmicron\u003c/sub\u003e-ACE2 and RBD\u003csub\u003eDelta\u003c/sub\u003e-ACE2 complex in NPT essemble. The aMD technique is used to enhance sampling in the protein\u0026apos;s conformational space, artificially reducing the energy barriers that separate different states of a given system \u003csup\u003e53,54,61\u0026ndash;64\u003c/sup\u003e. Additionally, we used the Bio3D package \u003csup\u003e65\u003c/sup\u003e to perform the principal component analysis (PCA). The PCs were obtained from the diagonalization of the covariance matrix obtained from the Cartesian coordinates of the superposed C\u0026alpha; atoms of complex structure. To avoid an underestimate of the atomic displacement, an iterated superposition procedure was applied before the PCA, where residues displaying the largest positional differences were excluded at each round until only the invariant \u0026lsquo;core\u0026rsquo; residues remained\u003csup\u003e66\u0026ndash;69\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eProtein\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-protein binding free energy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we also evaluated the binding energy differences between the complexes and then the decomposition energy was added to assess the energy contribution of each amino acid during the binding of RBD to ACE2. The binding free energy for the each RBD-ACE2 complex was obtained using:\u003c/p\u003e\n\u003cp\u003e∆G\u003csub\u003ebind\u003c/sub\u003e = G\u003csub\u003eRBD\u0026minus;ACE2\u003c/sub\u003e - G\u003csub\u003eRBD\u003c/sub\u003e \u0026ndash; G\u003csub\u003eACE2\u003c/sub\u003e (1)\u003c/p\u003e\n\u003cp\u003eHere, G\u003csub\u003eRBD\u0026minus;ACE2\u003c/sub\u003e represent the average over the snapshots of a single trajectory of the MD RBD-ACE2complex, G\u003csub\u003eRBD\u003c/sub\u003e and G\u003csub\u003eACE2\u003c/sub\u003e corresponds to the free energy of RBD and ACE2 protein, respectively. The binding free energy was obtained using MMGBSA method \u003csup\u003e70,71\u003c/sup\u003e implemented in AMBER 16 \u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn order to calculate free energy with MMGBSA (Eq. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) 5000 frames were taken from the 10 ns of MD production using: \u003csup\u003e72\u0026ndash;74\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003eis important to note that the entropy contribution was not included in the calculations due to the difficulty of accurately calculating entropy for a large protein-protein complex. \u003csup\u003e75\u003c/sup\u003e It is also worth to note that the frames were taken from the most stable structure observed in PCA graphics.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eAnalysis of Molecular Dynamics of RBD-ACE2 complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll-atom aMD simulations allowed to explore the conformations of protein-protein complex over time for each system: RBD\u003csub\u003eWT\u003c/sub\u003e-ACE2, RBD\u003csub\u003ealpha\u003c/sub\u003e-ACE2, RBD\u003csub\u003eOmicron\u003c/sub\u003e-ACE2 and RBD\u003csub\u003eDelta\u003c/sub\u003e-ACE2 complex. \u0026nbsp;Figure 1 shows the RMSD during 200 ns of aMD for each system with respect to the reference structure of the equilibrium step. RBD\u003csub\u003eWT\u003c/sub\u003e-ACE2, RBD\u003csub\u003ealpha\u003c/sub\u003e-ACE2 and and RBD\u003csub\u003eDelta\u003c/sub\u003e-ACE2 complexes were within fluctuation in a range of 1 to 3 \u0026Aring; (Figure 1), while the RBD\u003csub\u003eOmicron\u003c/sub\u003e-ACE2 complex the present the different variation during simulation in a range of 1 to 4 \u0026Aring; (Figure 1). Therefore, the structural equilibrium was reached for all system (Figure 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn 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 RBD\u003csub\u003ealpha\u003c/sub\u003e residues show less fluctuation compared to the WT and its last variants (Delta and Omicron).\u003c/p\u003e\n\u003cp\u003eIn this study, we also explore the flexible region in protein-protein complex. through essential dynamics analysis. The\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003eFor 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 RBD\u003csub\u003eWT\u003c/sub\u003e and the RBD\u003csub\u003eomicron\u003c/sub\u003e variant present greater conformational fluctuations, however, the RBD\u003csub\u003ealpha\u003c/sub\u003e variant stands out for its greater stability. In PC1 there are not many movements in RBD and ACE2 (Figure 3). The main movement of RBD\u003csub\u003eWT\u003c/sub\u003e and RBD\u003csub\u003eomicron\u003c/sub\u003e 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBinding Free Energy MMGBSA and Decomposition by Residue\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 (∆G\u003csub\u003ebind\u003c/sub\u003e (MMGBSA)) based on the points of greatest stability of the aMD trajectory (see Table 1). The RBD\u003csub\u003eomicron\u003c/sub\u003e shows the highest binding affinity to ACE2, reflecting the infectivity process, but its conformational fluctuations is similar to the other variants. RBD\u003csub\u003eomicron\u003c/sub\u003e present an adaptive and non-aggressive process when compared to the RBD\u003csub\u003ealpha\u003c/sub\u003e (with free energy of binding equal to -62.7836 kcal/mol), which demonstrated the lower free energy than RBD\u003csub\u003eWT\u003c/sub\u003e (-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.\u003c/p\u003e\n\u003cp\u003eThe RBD\u003csub\u003eDelta\u003c/sub\u003e has a higher binding affinity with the human receptor compared to the RBD\u003csub\u003eWT\u003c/sub\u003e (-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 ∆G\u003csub\u003ebind\u003c/sub\u003e in comparison to RBD\u003csub\u003eWT\u003c/sub\u003e. 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e: Binding free energy for native systems (SARS-CoV-2) and variants (Alpha/Delta/Omicron).\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEnergy (kcal/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eWT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAlpha\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDelta\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOmicron\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e∆E\u003csub\u003evdw\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-95.6(0.18)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-107.3(0.21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-103.4(0.16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-96.4(0.16)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e∆Eele\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-625.8(0.94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-608.5(0.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-955.1(1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-1381.7(1.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e∆E\u003csub\u003eGB\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e675.0(0.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e667.5(0.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1006.3(1.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1416.2(1.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e∆E\u003csub\u003esurf\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-13.4(0.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-14.5(0.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-13.9(0.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-13.5(0.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e∆G\u003csub\u003egas\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-721.3(0.96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-715.8(0.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-1058.5(1.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-1478.1(1.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e∆G\u003csub\u003esol\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e661.6(0.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e653.0(0.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e992.4(0.99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1402.7(1.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e∆G\u003csub\u003ebind (MMGBSA)\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-59.7(0.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-62.8(0.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-66.1(0.21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-75.4(0.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of mutations can be investigated through the free energy calculations that track the influence of changes in certain positions \u003csup\u003e76\u003c/sup\u003e. The results of the energy of decomposition by residue for RBD\u003csub\u003eWT\u003c/sub\u003e-ACE2, RBD\u003csub\u003eAlpha\u003c/sub\u003e-ACE2, RBD\u003csub\u003eOmicron\u003c/sub\u003e-ACE2 and RBD\u003csub\u003eDelta\u003c/sub\u003e-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 RBD\u003csub\u003eOmicron\u003c/sub\u003e provide favorable interaction between RBD\u003csub\u003eOmicron\u003c/sub\u003e and ACE2. Curiously, all these mutations include positively charged residues Lys or Arg (see Table 2). For example, K478 in RBD\u003csub\u003eOmicron\u003c/sub\u003e present a stabilization effect (-85.8 kcal/mol), while T478 in RBD\u003csub\u003eWT\u003c/sub\u003e 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe N501Y mutation in the RBD\u003csub\u003eAlpha\u003c/sub\u003e 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 RBD\u003csub\u003eAlpha\u003c/sub\u003e to ACE2, compared to the RBD\u003csub\u003eWT\u003c/sub\u003e, 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. \u0026nbsp;The mutations present in RBD\u003csub\u003eOmicron\u003c/sub\u003e demonstrate that during the gain in the energetic contribution of the residues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSome 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).\u003c/p\u003e\n\u003cp\u003eTable 2: Decomposition energies per residue in kcal/mol for the main mutation positions of RBD WT, Alpha, Delta and Omicron.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSARS-CoV-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAlpha\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDelta\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOmicron\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eG339\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eG339D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e68.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS371\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS371L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS373\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS373P\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS375\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS375F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eK417\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-121.2\u003c/p\u003e\n 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\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eQ498R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-161.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eN501\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eN501Y\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-10.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eN501Y\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eY505\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eY505H\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we evaluated the effect of residues mutation on structural and energetics of Spike protein RBD from SARS-CoV-2 variants in complex with the human ACE2 receptor. All-atoms accelerated Molecular Dynamics simulations and PCA analysis shows that that the RBD\u003csub\u003eOmicron\u003c/sub\u003e-ACE2 complex present similar fluctuation in comparison to S protein from Native, Delta and Alpha variants. The binding affinity of each RBD\u003csub\u003ex\u003c/sub\u003e to ACE2 was obtained using MM-GBSA methods. The results shows that the trend in the calculated binding free energies correlates well with virus infectivity of each variant. The mutation in RBD\u003csub\u003eOmicron\u003c/sub\u003e increase the affinity of Spike protein for ACE2 and may explain Omicron's high transmissibility in comparison with other SARS-CoV-2 variants. The stabilization effect RBD\u003csub\u003eOmicron\u003c/sub\u003e-ACE2 complex is achieved manly due the substitution of uncharged residues by positively charged residues: Lys and Arg in key positions. Overall, our results may explain at molecular level the effect of key mutations in the Spike protein for virus infectivity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll necessary files to conduct this work (.pdb and .parm7) can be found attached as the Supporting Information. The AMBER18 suite of programs and the Amber ff14SB force field were used to carry out the MD simulations and can found at https://ambermd.org/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) for their financial support. We also thank the access of the computational resources of the Supercomputer Santos Dumont (SDumont) provided by the Laborat\u0026oacute;rio de Computa\u0026ccedil;\u0026atilde;o Cient\u0026iacute;fica (LNCC), Apollo 2000 and CABANO \u0026ndash; UFPA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors contributed equally: Clauber Henrique Souza da Costa, Camila Auad Beltr\u0026atilde;o de Freitas.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWang, D. \u003cem\u003eet al.\u003c/em\u003e Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus\u0026ndash;Infected Pneumonia in Wuhan, China. \u003cem\u003eJAMA\u003c/em\u003e \u003cstrong\u003e323\u003c/strong\u003e, 1061\u0026ndash;1069 (2020).\u003c/li\u003e\n \u003cli\u003eHuang, C. \u003cem\u003eet al.\u003c/em\u003e Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e395\u003c/strong\u003e, (2020).\u003c/li\u003e\n \u003cli\u003eWho, W. 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Soc.\u003c/em\u003e \u003cstrong\u003e143\u003c/strong\u003e, 17646\u0026ndash;17654 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1401835/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1401835/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) variant Omicron spread more rapid than the other variants of SARS-CoV-2 virus. Mutations on the spike (S) protein receptor-binding domain (RBD) are critical for the antibody resistance and infectivity of the SARS-CoV-2 variants. In this study, we have used accelerated molecular dynamics (aMD) simulations and free energy calculations to present a systematic analysis of the affinity and conformational dynamics along with the interactions that drive the binding between Spike protein RBD and ACE2 receptor. We evaluate the impacts of the key mutation that occur in the RBDs Omicron and other variants in the binding with the human ACE2 receptor. The results shows that S protein Omicron have stronger binding to the ACE2 than other variants. The evaluation of the decomposition energy per residue shows the mutations N440K, T478K, Q493R and Q498R observed in Spike protein of SARS-CoV-2 provided a stabilization effect for the interaction between the SARS-CoV-2 RBD and ACE2. Overall, the results demonstrate that faster spreading of SARS-CoV-2 omicron may be correlated with binding affinity of S protein RBD to ACE2 and mutations of uncharged residues to positively charged residues such as Lys and Arg in key positions in the RBD.\u003c/p\u003e","manuscriptTitle":"Assessment of Mutations on RBD in the Spike Protein of SARS-CoV-2 Alpha, Delta and Omicron Variants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-17 18:36:35","doi":"10.21203/rs.3.rs-1401835/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-04-11T15:04:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-04-06T14:28:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"e74f93af-22c4-4aa4-91cb-eb5dbf14132a","date":"2022-03-30T04:45:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"973cfa00-f43e-4646-b6de-2dc293e5f8d2","date":"2022-03-25T11:52:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-03-22T11:51:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-03-21T12:37:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-03-16T09:05:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-03-16T09:02:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-02-27T22:20:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12d92ed2-f5ee-4faa-b6a0-c874a3ce8225","owner":[],"postedDate":"March 17th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-05-03T06:14:03+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-17 18:36:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1401835","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1401835","identity":"rs-1401835","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}