Probing the binding nature and stability of highly transmissible mutated variant Alpha to Omicron of SARS-CoV-2 RBD with ACE2 via molecular dynamics simulation

doi:10.21203/rs.3.rs-2213388/v1

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Probing the binding nature and stability of highly transmissible mutated variant Alpha to Omicron of SARS-CoV-2 RBD with ACE2 via molecular dynamics simulation

https://doi.org/10.21203/rs.3.rs-2213388/v1

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As of now there is no approved drug available as a causative agent of COVID-19. To resolve this issue, quickly identify a repurposed drug and develop new vaccines are the most promising solutions as it is the time-consuming process to design a new drug. As a therapeutic development, some vaccines and some repurposed drugs were approved to prevent people from SARS-CoV-2 infections during this pandemic period; all these medicines are made available based on the first structure of SARS-CoV-2 reported in late 2019 from Wuhan, China. Since then, vividly it is known that due to serious mutations in this virus, often its structure has been modified and emerged as the new types of variants; evidently, the new variants are the most dangerous and spreading very fast. SARS-CoV-2 variants are becoming the significant challenge to the current pandemic situation. Hence, the detailed structure of these variants is necessary to design new drugs and vaccines to inhibit the mutated SARS-CoV-2 variants. Present study analyzed the binding mode of receptor binding domain (RBD) of SARS-CoV-2 with the human angiotensin converting enzyme 2 (ACE2) using molecular dynamics simulation up to 500 ns. Notably, in some variants found an unusual binding mode of RBD with ACE2 forming different interactions; this was confirmed from the comparison of interaction between RBD-ACE2 of all variants with the wild type structure. Binding free energy values confirm that they exhibit high binding affinity towards ACE2. From this analysis, it is confirmed that the variations in the sequence of SARS-CoV-2 spike protein altered the binding mode which led to the high transmissibility caused new infections. This in-silico study on mutated variants alpha to omicron of SARS-CoV-2 RBD with ACE2 insights their binding mode, binding affinity and stability. This information is useful to design potential drug and vaccine to cure the devastating disease COVID-19.

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Covid-19 is an infectious disease, which was first reported in Wuhan, china on December 2019 and created high pandemic situation in worldwide. This disease is caused by severe acute respiratory syndrome Corona virus 2 (SARS-CoV-2) and it is called as corona virus disease 2019 (Covid-19). As of September 2022, globally 65.01 lakh people were died due to covid-19 (https://covid19.who.int/). To date there is no clinically approved drug is available to cure Covid-19. To fight against this disease, vaccination developments and repurposed drugs are the immediate solution to resolve this problem. Moreover, the main challenge in the current pandemic situation is, facing the newly generating different variants of SARS-CoV-2 virus, accordingly design new vaccines and drugs, and gain immunity for the affected people. SARS-CoV-2 virus is a single-strand RNA virus, and the genome encodes four structural proteins such as Spike (S) protein, Small Envelope (E) protein, Membrane (M) protein, and Nucleocapsid protein. Spike protein is a type I fusion protein that builds trimmers on virion surface. It has two subdomains such as S1 and S2; S1 domain is responsible for receptor binding which includes RBD and S2 domain is for membrane fusion¹. Currently, spike (S) protein is undergoing different structural mutations, and it is very important to investigate its structural and biological significance of mutations². Viruses are regularly changing their structure called as mutation. When a virus has one or more mutations on its structure is known as the variant of wild type/original virus. In viruses, generally the mutations occur in their surface proteins; this can significantly modify the viral infection function as well as the interactions with the neutralizing antibodies. In RNA viruses, the rate of mutations are higher than the DNA viruses; mutations are the significant building blocks of evolution of new infections^3,4. During the infection, SARS-CoV-2 RBD of S-protein binds with the human ACE2 protein form SARS-CoV-2 RBD–ACE2 complex. Different mutations in SARS-CoV-2 RBD cause new infections when they bind with ACE2. Understanding the structure and conformation of this RBD-ACE2 complex, and the interactions of different mutated SARS-CoV-2 RBD with the ACE2 are very crucial for the design of new drugs or vaccines. Mutated form of this virus emerges as a new form virus. In the present study, we have investigated the structure, interaction and the dynamic nature of reported different mutated forms of virus such as alpha to omicron SARS-CoV-2 RBD with ACE2 through in silico analysis. The SARS-CoV-2 RBD–ACE2 complex structure was reported and the coordinates and structure factor files are deposited in the Protein Data Bank (PDB); which is also similar to the previously reported SARS-CoV RBD–ACE2 complex structure^1,5,6. The evaluation of SARS-CoV-2 has been characterized by the development of mutations, in the context of ‘variants of concern’, that effective virus natures, including transmissibility and antigenicity, apparently in response to the immune profile modify the human population^7–9. The transmissibility and infecting nature of COVID-19 is relatively much higher than the SARS-CoV and MERS viruses, this made a situation of pandemic of respiratory syndromes in worldwide¹⁰. To understand the infections and pathogeneses nature of different variants of SARS-CoV-2 the in-vivo, in-vitro and clinical studies have been conducted^8,11–16 and still the same is also in progress. However, there is no detailed structural report about the dynamical nature and interactions of the alpha to omicron SARS-CoV-2 RBD-ACE2 complex of different SARS-CoV-2 variants. Importantly, the knowledge of dynamical binding nature of RBD with ACE2 is also essential to identify the drug targets for the variants which has mutations, this often alter the binding mode of virus and entry mechanism to the host cells. Several reports outline the evidence of conformational change of binding domain that occurs due to the mutations in SARS-CoV-2 variants^16–20. The present study explores the differences of the alpha to omicron variants (mutated) of SARS-CoV-2 RBD bound with human ACE2 from the analysis of their structure, intermolecular interactions and conformational modification using the molecular dynamics simulation and binding free energy studies; and the results of all the mutated complexes are compared with the wild type structure.

Protein preparation

The crystal structure of the complex form of SARS-CoV-2 S-protein RBD with ACE2 has been downloaded from the protein data bank (PDB Code: 6M0J)⁶. As per the variants data reports in corresponding RBD domain sequences (333-526), the wild type residues were replaced with mutated residues by using residue mutate option in Maestro panel. The mutated RBD residues are B.1.1.529 (G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H), B.1.617 (L452R and E484Q), B.1.617.2 (L452R and T478K), B.1.617.2.1 (K417N), B.1.618 (E484K), B.1.1.7 (N501Y), B.1.351 (K417N, E484K, and N501Y), and P.1 (K417T, E484K, and N501Y). The mutated complexes were submitted to the protein preparation using protein preparation wizard which is included Epik module of Schrödinger Maestro⁴¹. In the initial step of protein preparation, the structure of variants has been pre-processed by Prime module to predict the protein structures and proceed for optimization. Further, the optimized protein structures were energy minimized with OPLS4 force field⁴².

Molecular dynamics simulations

The energy minimized protein structures are subjected for molecular dynamics (MD) simulation to understand their conformational stability, intermolecular interactions, binding mode and binding energy. In Schrödinger Maestro package, Desmond system builder module was used to prepare the complex of alpha to omicron variant of SARS-COV-2 RBD with ACE2 for MD simulation. In this process, for each complex a predefined TIP3P orthorhombic 10x10x10 Å water box was chosen as solvent model⁴³. To neutralize each complex variant, the Na⁺/Cl^- ions were added into the TIP3P solvent box and all the MD systems were prepared with OPLS4 force field for MD simulation. Further, the MD simulation was performed for each wild and mutated complex with NPT ensembles at the temperature 310 K and 1 atm pressure with the time scale of 500 ns using Desmond MD package module incorporated with the Schrödinger⁴⁴.

Binding free energy

In biological system, most of the biological activities are regulated by the intermolecular interactions between the proteins components present in each cell. These intermolecular interactions play an important role in the protein-protein contact; the strength of these interactions can be estimated from the binding free energy of protein-protein interactions. In the present study, the binding free energy of RBD-ACE2 for wild and all mutated complex of alpha to omicron have been calculated. PRODIGY online (PROtein binDIng enerGY) web server was used to calculate the binding free energy (ΔG) values⁴⁵ reported here. The binding free energy was calculated between RBD and ACE2 of all the variants which we have taken to study, in both energy minimized state (after mutation) and MD simulation state. And also these results were compared with the wild type to understand its binding affinities.

Intermolecular interactions of wild and mutated SARS-CoV-2 RBD with ACE2: The MD simulations were performed for wild and mutated SARS-CoV-2 RBD with ACE2 complex B.1.1.7-alpha, B.1.351-beta, B.1.617-delta, B.1.617.2-delta, B.1.617.2.1-delta plus, B.1.618, and P.1-gamma, and B.1.1.529-omicron. The mutation in SARS-CoV-2 RBD altered the binding mode of the virus with the human ACE2 protein, this leads to the change of transmissibility of virus into the human body. On comparing the interactions and the binding energy of each mutated variant of the SARS-CoV-2 RBD - ACE2 complex with the wild SARS-CoV-2 RBD-ACE2 complex allows to understand the binding nature and dynamics of mutant RBD-ACE2 complex. This allows to predict the variability of transmission of differently mutated RBD-ACE2. In the present study, the MD simulation was performed for each RBD-ACE2 complex up to 500 ns.

Wild type: Interactions between the wild SARS-CoV-2 RBD and ACE2: In prior to the MD simulation, the wild SARS-CoV-2 RBD with ACE2 complex was energy minimized. Further, the MD simulation was performed at 500 ns time scale for the wild SARS-CoV-2 RBD with ACE2 complex. The interactions of MD simulated wild complex have compared with the corresponding energy minimized complex of wild complex. The MD simulation shows that, some of the interactions exist between RBD and ACE2 in the initial state of RBD-ACE2 complex are become more stable during the simulation and the interactions are Asn487∙∙∙Gln24, Tyr489∙∙∙Tyr83, Thr500∙∙∙Asn330, Gln498∙∙∙Lys353, and Gly502∙∙∙Lys353 and the interaction distances are 1.94, 1.86, 3.03, 1.82 and 1.84 Å respectively (energy minimized state) as shown in figure 1. During the MD simulation, the above interactions were maintained; the corresponding distances in the final frames are 1.98, 1.86, 3.02, 2.0, and 2.06 Å respectively. And these interactions are also comparable with the reported complexes⁶ by Jun Lan et al,.

Alpha Variant (B.1.1.7): Interactions between alpha variant of SARS-CoV-2 RBD and ACE2: The recent report outlines that there are nine modifications among the amino acids of S-protein of alpha variant, this includes the deletion of 69-70 and 144, and the mutants N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H^22,23. Notably, the mutation of N501Y residue within the RBD shows enhanced binding affinity towards the murine and human ACE2 receptor^1,14. The energy minimized mutated RBD-ACE2 complex shows that the RBD forms hydrogen bonding interactions with ACE2 (Figure 2) and the interactions are Asn487∙∙∙Tyr83, Gly496∙∙∙Lys353, Asn487∙∙∙Gln24, and Tyr489∙∙∙Tyr83; these interactions are largely different from the interactions found in wild type. However, the Tyr489∙∙∙Tyr83 interaction found in wild type also present in this alpha variant B.1.1.7. However, this interaction was disappeared during the MD simulation and resulted with the interactions Thr333∙∙∙Glu589, Glu340∙∙∙His239 and Lys444∙∙∙Asp609, the corresponding interaction distances are 2.08, 1.84, 1.95 Å respectively. Overall, it indicates that the above said mutations are significantly modified the binding mode of RBD of S-protein with the ACE2. This could be the reason that the alpha variant B.1.1.7 has high transmission ability into the human body.

Beta variant (B.1.351) - Interactions between the beta variant of SARS-CoV-2 RBD and ACE2: This variant was identified in South Africa and it is called as beta variant. This variant has some deletions and substitutions in S-protein, namely D80A, ΔL242-244, K417N, E484K, N501Y, D614G and A701V. Beta variant is the mutated form of the wild type SARS-CoV-2 RBD. In the present study, the MD simulation was performed for this mutated RBD-ACE2 complex. In prior to the MD simulation, the complex has been energy minimized. In the energy minimized mutated RBD-ACE2 complex, the RBD forms interactions with ACE2 (Figure 3) and the interactions are Asn487∙∙∙Tyr83, Gly496∙∙∙Lys353, Tyr449∙∙∙Asp38, Tyr489∙∙∙Tyr83, Gly502∙∙∙Lys353, the interaction distances are 1.64, 2.68, 1.83, 1.86, 1.94 Å respectively. Among these interactions, notably, some of them are same as found in the wild type. As found in wild type, the Asn501 residue forms interactions with Lys353 and beta mutant (N501Y) at the distance 2.49 and 1.97 Å respectively. During the MD simulation, the interactions in the complex have been modified in different binding regions, and the new interactions are Thr333∙∙∙Glu589, Asn334∙∙∙Thr593, and Glu340∙∙∙Lys596 and the corresponding distances are 1.96, 2.23, and 2.58 Å respectively. These interactions are neither comparable with wild type nor with the above energy minimized complex; perhaps, this difference may be attributed to the large motion and conformational modification of amino acids of binding region during the MD simulations; this leads the RBD to form new interactions with ACE2. However, the presence of the above said mutations in RBD provides tight binding (interactions) with ACE2 and this facilitates for widespread escape from monoclonal antibody neutralization. The previous results indicate that the B.1.351 may increase the risk of disease infection among immunized individuals^{23, 27}.

Delta variant (B.1.617) - Interactions between the delta variant of SARS-CoV-2 RBD and ACE2: The variant B.1.617 is known as Indian variant, which was first identified in India. The S-protein substitutions of this variant are L452R, E484Q and D614G. The main attribute of this variant is, potential neutralization reduction by some monoclonal antibody treatments and reduced neutralization by the post vaccinations, although an increase in breakthrough infections may arise as a result of the reduced neutralizing capacity of sera^28-31. This Indian variant is also known as double mutant, which is mutated in the RBD region of S-protein. Before perform the MD simulation, the structure of this mutated RBD with ACE2 complex was energy minimized. In the energy minimized complex, the RBD forms several interactions with ACE2, some of the interactions are Lys417∙∙∙Asp30, Thr500∙∙∙Tyr41, Gln498∙∙∙Lys353, Asn501∙∙∙Lys353, Tyr449∙∙∙Asp38, Asn487∙∙∙Gln24, Gln498∙∙∙Asp38, Gly502∙∙∙Lys353, and Tyr505∙∙∙Glu37, and these are very strong, the corresponding distances are 1.98, 2.69, 2.35, 3.03, 1.78, 2.27, 2.21, 1.97, and 2.01 Å respectively. Further, the MD simulation was carried out for this mutated RBD-ACE2 complex. Surprisingly, all the above said interactions are intact even after the MD simulation, however the distances are slightly modified, the new interaction distances are 2.65, 1.98, 2.67, 2.99, 1.87, 2.05, 2.28, 1.62, and 1.85 Å respectively. Among these, notably, the Asn487∙∙∙Gln24 and Gly502∙∙∙Lys353 interactions are similar to the interactions found in the wild type RBD-ACE2 complex as well as the minimized state of the mutated structure (Figure 4); interestingly, these interactions are highly stable during the MD simulation.

Delta variant (B.1.617.2) - Interactions between this delta variant of SARS-CoV-2 RBD and ACE2: The variant B.1.617.2 is also an Indian variant and it is another delta variant exhibits different substitutions in the S-protein, namely T19R, G142D, R158G, L452R, T478K, D614G, P681R and D950N, and the deletion of E156 and F157. Notably, this variant has some attributes, such as increased transmissibility, reduced neutralization by some post vaccination sera and monoclonal antibody treatments²³. The in vitro studies reveals that the influence of the delta variant has been mostly directed by a combination of evasion of neutralising antibodies in the previously infected persons and increased virus infectivity^12,31-33. The results of MD simulation studies of mutated RBD-ACE2 complex (Figure 5) shows that the Arg346∙∙∙Glu483, Lys444∙∙∙Asp609, Asn343∙∙∙Ser602 and Thr345∙∙∙Val604 interactions exist between RBD and ACE2 and are found strong, the corresponding interaction distances are 1.84, 2.06, 1.86, and 1.66 Å respectively. Due to mutation in the in the RBD, the interacting amino acids with the ACE2 of wild complex is different; furthermore, during the MD simulation large conformational modification took place in both RBD and ACE2 regions, this can be well understood when compare with the results of the above said energy minimized delta variant RBD-ACE2. And it is also noteworthy that the mutated residues L452R and T478K are not forming any interaction with the ACE2.

Delta plus variant (B.1.617.2.1)-Interactions between the delta plus variant of SARS-CoV-2 RBD and ACE2: The variant B.1.617.2.1 (delta plus/AY.1) also an Indian variant, which was identified in India. This variant exhibit one main mutation in S-protein known as K417N and particularly this mutation is also found in the RBD region and this is mutated from the Indian variant B.1.617.2. Furthermore, this variant was characterised as reduced potency for vaccinations and it is highly infectious. The results of MD simulation of mutated RBD-ACE2 complex reveals that RBD forms strong Tyr489∙∙∙Tyr83, Gln493∙∙∙Glu35 and Thr500∙∙∙Tyr41 types of interactions with ACE2, the interaction distances are 1.77, 1.94 and 1.97 Å respectively (Figure 6). Interestingly, these interactions are also found in the energy minimized complex, where the interaction distances are 2.58, 2.04, and 1.80 Å respectively; this confirms that during the MD simulation these interactions are intact and the distances are not much altered. Whereas with the wild type, the only Tyr489∙∙∙Tyr83 interaction is commonly found and no other interaction is comparable. The results of MD simulation indicate that this mutated RBD forms closer interactions with the neighbouring amino acids of ACE2.

Triple mutant (B.1.618) - Interactions between the variant Triple mutant SARS-CoV-2 RBD and ACE2: The variant B.1.618 is the triple mutant, which was identified in India, the mutations present in the S-protein are the deletion of Y145, H146, E484K and D614G³⁰. In the RBD region, there is only one residue has been mutated. The variant B.1.618 S-protein is neutralized with 2-5 fold cutback by recovering sera and vaccine-elicited antibodies^31,32. Prior to the MD simulation, in minimized state, for B.1.618 variant, the intermolecular interactions are formed as obtained from the wild type with some less distance differences. The MD simulation reveals that the RBD forms Glu340∙∙∙His239, Asn343∙∙∙Val604, Asn334∙∙∙Leu585, Asn343∙∙∙Lys600, and Thr345∙∙∙Val604 (Figure 7) interactions with ACE2 at the distances 1.98, 1.79, 1.88, 1.86, 2.26 Å respectively. These interactions are not comparable with the wild type RBD-ACE2 complex and are different.

Gamma variant (P.1) - Interactions between the variant Gamma variant SARS-CoV-2 RBD and ACE2: The variant P.1 is also known as gamma variant, which was identified in Brazil and Japan initially, the mutations presented as L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T1027I in the S-protein³⁸. In the RBD region, E484K only has been mutated. For P.1 variant the intermolecular interactions are formed similar to the wild type at minimized state. The MD simulation shows that the interaction between RBD and ACE2 are Glu340∙∙∙Lys596, Arg346∙∙∙Asp609, Asn334∙∙∙Asn586, Thr345∙∙∙Val604, and Tyr449∙∙∙Asp609 at the distances 1.96, 2.01, 2.13, 2.64 and 1.84 Å respectively (Figure 8). As these interactions totally found with new binding sites of the proteins, they are not comparable with the wild type RBD-ACE2 complex. These new interactions are formed due to the changes between binding sites of RBD and ACE2 during the MD simulation.

Omicron - Interactions between the omicron variant of SARS-CoV-2 RBD and ACE2: In the omicron variant, the mutated residues of S-protein RBD are G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H. Due to high mutation, the mode of interaction of omicron RBD with ACE2 is highly altered. This can be well understood from the interactions of energy minimized RBD-ACE2 complex of Omicron variant. The RBD bounds with the ACE2 forms hydrogen bonding interactions with ACE2 (Figure 9) are Lys31∙∙∙Arg493, Glu35∙∙∙Arg493, Asp38∙∙∙Tyr449, Asp38∙∙∙Ser496, Asp38∙∙∙Arg498, Tyr83∙∙∙Asn487, Asn330∙∙∙Thr500, Lys353∙∙∙Gly502, Lys353∙∙∙His505, Asp355∙∙∙Thr500 with the distance of 2.81, 2.19, 2.04, 2.3, 1.87, 2.0, 2.26, 2.15, 2.15, 2.4 Å respectively. Further, to understand the stability of these interactions in the RBD-ACE2 complex, the MD simulation was carried out and the intermolecular interactions also analysed. The amino acid residues of RBD forms hydrogen bonding interactions with the residues of ACE2, the interactions are Lys596∙∙∙Glu340, Phe603∙∙∙Asn343, Val604∙∙∙Ala344, Val604∙∙∙Thr345, Tyr613∙∙∙Asp442 and Tyr613∙∙∙Lys444 with the distance of 2.06, 2.74, 2.44, 2.49, 1.58 and 1.87 Å respectively. On comparing these interactions with the above energy minimized complex reveals that during the MD simulation, a large conformational modification took place in both RBD and ACE2 proteins; this effect altered the binding mode of S-protein and resulted new interactions. Furthermore, these interactions also not comparable with the wild type of SARS-CoV-2 RBD – ACE2 complex as the interactions are different.

RMSD analysis of Wild type and mutation variants:

The root means square deviation (RMSD) of ligand and proteins in the ligand-protein complex illustrates their deviations during the MD simulation. Figure 10 shows the variation of RMSD values of wild type and all the mutation variants complexes during the MD simulations up to 500 ns (Table 1). For wild type, the average RMSD range is 2.0 to 4.0 Å. Notably, during the 225 to 270 ns period of MD simulation, the RMSD peaks are found in the range of 3.2 to 6.6 Å.

The MD simulation of B.1.1.529 (Omicron) shows the variation of RMSD in the range of 1.8 to 4.2 Å, in which, up to 60 ns the deviations maintained in the range 1.8 to 3.0 Å, beyond that the RMSD slightly increased up to 4.7 Å and this trend was found up to 130 ns. Further, the whole remaining fickle peaks of RMSD range from 2.7 to 3.7 Å and this trend maintained up to 500 ns. Notably, the variation RMSD of omicron is similar to the wild type, and no significant deviation is found, this confirms that the omicron RBD-ACE2 complex is stable during the entire MD simulations.

In B.1.617 (Indian variant/double mutant), the RMSD variation is found in the range of 2.5 to 3.5 Å. During the simulation, some deviations are noticed, however they are not very significant. In B.1.617.2 (Indian/Delta variant) up to 200 ns, the RMSD value is slightly increased from 1.8 to 4.8 Å and further some fickle peaks were found until 500 ns simulation and are normal. Overall, the variation of RMSD ranges from 2.6 to 4.8 Å. Whereas in B.1.617.2.1 (Indian/Delta plus variant) the variation of RMSD also found up to 150ns during the MD simulation. Initially, the values were in the range 1.8 to 2.5 Å this exist up to 85 ns, after that a sort while the RMSD was increased to 4.5 Å. Further, this value fluctuated and not exceeded the RMSD beyond 4.2 Å and remained maximum of same until 500 ns. Indian variant B.1.618 (Indian variant/double mutant) has the average variation of RMSD value in the range 2.0 to 4.0 Å up to 500 ns. However, there is no notable deviation is found and remained normal and stable.

In the RMSD plot of B.1.1.7 (UK variant), there are some fickle peaks are appeared during the 500 ns MD simulation. The variation of RMSD during the simulation is in the range 2.4 to 3.6 Å, which is in the acceptable level. Whereas in B.1.351 (South African) variant, initial part of simulation up to 70 ns, the RMSD value remained to 2.4 Å. After that, up to 500 ns of simulation, the RMSD value is found in the range 3.0 to 4.3 Å. In P.1 (Brazil variant) up to 150 ns, the RMSD is in the range of 1.6 to 3.2 Å; beyond that the RMSD value increased to in the range of 3.2 to 4.8 Å and this trend remained until 500 ns.

RMSF analysis of wild type and mutation variants:

Figure 11 shows the RMSF plots of wild type and all the mutated variants of SARS-CoV-2 RBD – ACE2 complexes (Table 2). Among these, notably, a high fluctuation is found in the loop region of wild type RBD-ACE2. Particularly, the RMSF of RBD residues Asp136 (6.59 Å), Asp157 (5.90 Å) and ACE2 residues Thr333 (5.57 Å), Ser371 (4.87 Å) and His519 (4.89 Å) shows high fluctuations which are found in the loop regions. Whereas in the other variants, similar trend is also found notably in the loop regions residues have high fluctuations. On compare with the loop regions, the fluctuation of helix and sheet residues are relatively less. However, the average RMSF fluctuation is found in the range of the variants are within 0.8 to 3 Å. From these results, it is confirmed that no significant RMSF variations is found in all reported variants in sheets and helix region residues whereas loop region residues exhibit large fluctuations.

Binding free energy calculations

The binding free energy values (ΔG) allow to understand the binding affinity of variants with ACE2. The binding energy of all the variants with ACE2 has been calculated and the results are compared with the wild type RBD-ACE2 complex (Table 3). The ΔG values are calculated from the final frames of the 500 ns scale of MD simulations and the energy minimized complexes of all mutant variants and wild type RBD-ACE2 complex reported here. The calculated ΔG value for the wild type is -11.7 kcal/mol, whereas for the mutant variants B.1.617, B.1.617.2, B.1.617.2.1, B.1.618, B.1.1.7, B.1.351, P.1, and B.1.1.529, the values are -10.7, -8.5, -11.6, -11.7, -8.2, -5.8, -7.3, and -9.1 kcal/mol respectively. Relatively, the binding energy ΔG of wild type found to be similar to all variants and the same trend is also observed in all energy minimized RBD-ACE2 complexes of the variants.

Table 3: Binding free energy (ΔG) values (kcal/mol) of wild and other selective variants of SARS-CoV-2 RBD-ACE2.

Name of the variants		ΔG (kcal/mol)
	0 ns		250ns	500 ns
Wild	-12.5		-11.3	-11.7
B.1.1.529 (Omicron)	-10.5		-9.1	-9.1
B.1.617 (Indian Variant/Delta)	-11.9		-11.4	-10.7
B.1.617.2 (Delta)	-11.9		-9.9	-8.5
B.1.617.2.1 (Delta plus)	-12.2		-11.6	-11.6
B.1.618 (Indian Variant)	-11.7		-8.0	-11.7
B.1.1.7 (UK Variant)	-12.6		-7.0	-8.2
B.1.351 (South African Variant)	-12.2		-11.5	-5.8
P.1 (Brazil Variant)	-11.8		-9.4	-7.3

Hence, we can understand that from these ΔG values, some of the mutant variants sustain the affinity with less differences as presented in minimized state after the MD simulation (Figure 12). As well as, some of the variants shows less affinity in MD simulation than presented in minimized state. However, the overall results confirm that SARS-CoV-2 RBD is binding with ACE2 with good binding affinity. Besides, according to the calculated ΔG values, B.1.617, B.1.617.2, B.1.617.2.1, and B.1.618 exhibits high binding affinity than other variants (B.1.1.7, B.1.351and P.1) due to its less energy values.

This in-silico study has been performed to understand the binding nature of some SARS-CoV-2 RBD variants with ACE2. Among the variants of concern, the present dynamics simulation found that both B.1.617 and B.1.617.2.1 variants exhibit almost same RBD-ACE2 binding interactions and are comparable with the wild complex. Whereas, in the other variants (B.1.1.529-omicron, B.1.1.7-alpha, B.1.351-beta, B.1.617.2-delta, B.1.618 and P.1-gamma), the RBD binds at different locations (adopts different binding domain) of ACE2 protein; in consequence of that the interactions between RBD and ACE2 are found different; however, some interactions are found common. The binding free energy analysis of B.1.617 (delta), B.1.617.2.1 (delta plus), B.1.618 (triple mutant) shows high binding affinity compare with other variants similar to the wild type (< -10 kcal/mol). This may be the main reason for the inactivity of newly designed vaccines or repurposed drugs against the target, which was initially reported as a binding site of the spike protein. Further RMSD and RMSF analysis shows that in all the variants, the complexes are very stable during the MD simulation. The results of this in silico study suggest that during the MD simulations, some of the RBD variants bind with ACE2 at different locations; these new binding sites can be considered as new target sites as already reported as binding sites in the drug discovery process. Based on the results of this in silico study, the suggested new binding sites in ACE2 may be further confirmed by experimental studies to design new drug to effectively treat COVID-19.

Acknowledgments

The authors thank the Computer Centre, Periyar University, Salem, for performing the computational work in the High Performance Cluster (HPC) Computer.

Authors contributions

R.J and P.K designed the research work and did the MD simulation works; R.J, C.A and S.S performed the analysis. All authors equally contributed to preparing and reviewing the manuscript.

Competing interests

The authors declare no competing interests.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Tables 1-2 is available in the Supplementary Files section.

No competing interests reported.

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Probing the binding nature and stability of highly transmissible mutated variant Alpha to Omicron of SARS-CoV-2 RBD with ACE2 via molecular dynamics simulation

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