Investigation of Critical Binding Pattern in SARS-CoV-2 Spike Glycoprotein with Angiotensin-Converting Enzyme 2: An in Silico Analysis 


 Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a newly-discovered coronavirus and responsible for the spread of coronavirus disease 2019 (COVID-19). SARS-CoV-2 infected millions of people in the world and immediately became a pandemic in March 2020. SARS-CoV-2 belongs to the beta-coronavirus genus of the large family of Coronaviridae. It is now known that its surface spike glycoprotein binds to the angiotensin-converting enzyme-2 (ACE2), which is expressed on the lung epithelial cells, mediates the fusion of the cellular and viral membranes, and facilitates the entry of viral genome to the host cell. Therefore, blocking the virus-cell interaction could be a potential target for the prevention of viral infection. The binding of SARS-CoV-2 to ACE2 is a protein-protein interaction, and so, analyzing the structure of the spike glycoprotein of SARS-CoV-2 and its underlying mechanism to bind the host cell receptor would be useful for the management and treatment of COVID-19. In this study, we performed comparative in silico studies to deeply understand the structural and functional details of the interaction between the spike glycoprotein of SARS-CoV-2 and its cognate cellular receptor ACE2. According to our results, the affinity of the ACE2 receptor for SARS-CoV-2 was higher than SARS-CoV. According to the free energy decomposition of the spike glycoprotein-ACE2 complex, we found critical points in three areas which are responsible for the increased binding affinity of SARS-CoV-2 compared with SARS-CoV. These mutations occurred at the receptor-binding domain of the spike glycoprotein that play an essential role in the increasing the affinity of coronavirus to ACE2. For instance, mutations Pro462Ala and Leu472Phe resulted in the altered binding energy from -2 kcal·mol−1 in SARS-COV to -6 kcal·mol−1 in SARS-COV-2. The results demonstrated that some mutations in the receptor-binding motif could be considered as a hot-point for designing potential drugs to inhibit the interaction between the spike glycoprotein and ACE2.

Coronavirus particles have a helical nucleocapsid structure, consisting of the viral genomic RNA and nucleocapsid (N) phosphoproteins, which is surrounded by a lipid bilayer. The RNA genome of the virus contains about 30000 nucleotides and produces 3 or 4 types of structural proteins, including membrane (M), spike (S), and envelope proteins (E), as well as the hemagglutinin-esterase (HE) protein, which is detected in some types of coronaviruses 3 . By producing adequate quantities of novel structural proteins and genomic RNA, the particles are assembled. The assembly and release of virions are the nal steps of the viral life cycle.
The spike glycoprotein (also called protein S) has a key role in viral attachment, entry, and fusion and serves as a target for developing the producing antibodies, vaccines, and designing the potential inhibitors 4 . The protein S is produced as a precursor that contains almost 1,300 residues; then cleaved to two subunits; 1-a carboxyl (C)-terminal (S2 subunit) region and an amino (N)-terminal (S1 subunit) region. A trimer spike is synthesized and exposed on the viral envelope by assembling three S1/S2 heterodimers. A receptor-binding domain includes an entry S1 subunit mediating viral entry into the host cells through binding to the host receptor. Also, the S2 subunit contains two heptad repeat areas that participate in the fusion process 5 .
It has been demonstrated that different receptors are bounded to RBDs of MERS-CoV and SARS-CoV.
Angiotensin-converting enzyme 2 (ACE2) is one of the important receptors that can bind to SARS-CoV; while, dipeptidyl peptidase-4 binds to MERS-CoV. ACE2 is an enzyme with 802 residues that is expressed on the surface of the cell membrane of several tissues (lungs, arteries, heart, kidney, and intestine) and interacts with the spike glycoprotein in some coronaviruses, including HCoV-NL63, SARS-CoV, and SARS-CoV-2. ACE2 has two domains in the extracellular region, including the zinc metallopeptidase domain (residues 19-611) and the C-terminal domain (residues 612-740). The zinc metallopeptidase domain is composed of three regions that interacts with SARS-CoV spike glycoprotein through residues positioned at 30-41, 82-84, and 353-357 7 ACE2 also binds to SARS-CoV-2 through the S protein expressed on the surface of the virus. Thus, it is vital to deeply investigate the RBD expressing on SARS-CoV-2 S as a potential target for developing the potent inhibitors, designing the vaccines, and production of neutralizing antibodies 8 . The binding a nity of ACE2-spike is crucial for SARS-CoV-2 infection e ciency and completely dependent to the structure and interaction pattern of spike glycoprotein form SARS-CoV-2. Since the binding domain from each structures is available, so it is possible to measure the a nity of the whole complex 9 .
Herein, we investigated the interaction pattern of ACE2-Spike protein complex using in silico approaches to deeply understand the mechanism underlying the virus attachment and explain any potential differences in the binding pattern of SARS-CoV-2 and SARS-CoV to their cognate receptors to be able to propose new promising drugs with the highest e ciency for COVID-19 treatment. It seems that the inhibition of Spike-ACE2 interaction is the most straightforward and e cient method for the prevention of SARS-CoV-2 infection using peptides or small molecules. Previously, we have performed computational methods to understand the protein-protein interaction pattern at atomic levels to seek potentially speci c peptide inhibitors for cancer treatment 10,11 . In this study, several computational methods were utilized, including molecular dynamics simulation, MM-PBSA and interaction pattern analyses. The detailed molecular events could be explained by these methods from conformational alterations upon the interaction of the virus with its receptor to molecular interactions between the viral protein and the related receptors at atomic level. An intensive structural assessment was performed to investigate the interaction between the virus spike protein and its receptor.

Results
Three structure forms of ACE2-spike protein complexes, namely 2ajf, 6m0j, as well as a chimeric structure 6vw1 have been selected for the study. The chimeric structure is a receptor-binding domain of SARS-CoV that acts as a scaffold, while the receptor-binding motif of SARS-CoV-2 acts as a functional group for the interaction with ACE2 12 . Therefore, the comparison of three structures could be feasible to understand the role of spike mutations on the binding a nity to ACE2.

Analysis of spike (S) glycoprotein-ACE2 receptor interaction
At rst, three crystal structures (2ajf, 6m0j and 6vw1) were obtained, and then the differences in their structures and sequences were identi ed ( Figures S1 and S2). The results indicated that the majority of the mutations occurred in the receptor-binding motif of SARS-CoV-2 compared with SARS-CoV which is listed in the table S1. This motif plays an essential role in spike-receptor interaction. Then, we performed in silico comparison among these structures to deeply investigate the importance of mutations in the receptor-binding motif (RBM) and or other points of the receptor-binding domain (RBD) as well as the spike a nity to receptors to understand the binding of the virus to ACE2.
Also, a group of residues in the SARS-CoV-2 spike protein are present in the interface region of the four regions in ACE2 including Lys417, Gly446, Tyr449, Tyr453, Leu455, Phe456, Ala475, Phe486, Asn487, Tyr489, Gln493, Gly496, Gln498, Thr500, Asn501, Gly502, Tyr505. Ccomparative analysis of the RBD between SARS-CoV-2 and SARS-CoV showed the presence of several mutations in this area including Tyr442Leu, Leu443Phe, Leu472Phe, Asn479Gln, Tyr484Gln, and Thr487Asn. These mutations could be considered as important players in the binding sites of the spike protein in the ACE2 receptor, leading to an increase in binding a nity of SARS-CoV-2 to ACE2. For instance, mutation Tyr442Leu leads to change in interaction pattern between spike-ACE2 from SARS-CoV to SARS-CoV2. By means that, instead of residue 31, residue 34 of ACE2 is involved in virus-receptor interaction in the binding site which we will further discuss about this area. Accordingly, other mutations can also cause changes in the interacting pattern of SARS-CoV-2-ACE2 when compared with SARS-CoV. The detailed interaction patterns are shown in (Figure 1).

Analysis of the interacting region in the spike-ACE2 complex during MD simulation
PyContact was used for the analysis of non-covalent interactions between the spike and ACE2 receptor during the MD simulation to characterize the critical areas in ACE2 involved in the interaction with the spike. The contact areas between SARS-CoV and CoV-2-ACE2 with ACE2 receptor were depicted in gures S3 and S4. The obtained results showed ve and six regions of ACE2 interacted with SARS-CoV and CoV-2-ACE2 respectively and the highest contact area in both complexes were detected in residues 35-194 and 266-344. These two regions are not constantly involved in the interaction, but instead residues 35-50 and 300-330 have the highest participation in the interaction in ACE2. Also, analysis of the interface area demonstrated that a group of critical residues including residues 353-358 from region 346-372 of the ACE2 receptor interacted with the SARS-CoV-2 spike protein, while such interaction was not found in the SARS-CoV-ACE2 complex.
On the other hand, the interface region of the chimeric structure is completely different from two other structures (Fig. S5). Based on our results, there are seven regions in ACE2 that interact with chimeric structure. The chimeric structure has an interface region including 11 residues (495-505), which were nonspeci c for protein-protein interaction in the virus-receptor complex. In residues 19-33, the interface region has higher contact area than SARS-CoV-2 but lower than SARS-CoV. In three regions of ACE2 (residues 16-175, residues 224-246, residues 248-326), the interface area was about 800 Å 2 , while in two other regions (347-373 and 542-615) was about 300 Å 2 . Such a difference may stem from the integration of two regions from two different proteins that cause nonspeci c interactions.
Hydrogen bonds were analyzed in the four regions of the ACE2 receptor (19-33, 35-54, 325-331, and 334-339) and receptor-binding motif of the three spike structures during the simulation, and they are summarized in gure 2. Obtained results showed that the four regions of the ACE2 receptor participated in the interaction with the receptor-binding motif of the spike protein from SARS-CoV and SARS-CoV-2 (19-33, 35-54, 353-358, and 325-331).
The number of H-bonds in residues 19-33 of the receptor when interacting with the receptor-binding motif of the spike during the simulation was higher in complexes of chimeric structure and SARS-CoV-2 than that of SARS-CoV. Also, the number of H-bonds in residues 334-339 of the receptor in SARS-CoV-2 and chimeric structure complexes were more than that of SARS-CoV. However, the number of H-bonds in the spike-receptor complex in two regions 35-54 and 325-331 of SARS-CoV-2 and chimeric structure complexes were lower than SARS-CoV. In this way, the number of H-bonds in the four regions of the SARS-CoV-ACE2 complex when interacting with the receptor-binding motif of the spike protein was not the same as SARS-CoV-2 and chimeric structure complexes. Therefore, it appears that the number of Hbonds in the receptor-binding motif of SARS-CoV-2 when interacting with ACE2 was more than SARS-CoV.
These four regions of the receptor interact with speci c parts of the virus receptor-binding motif, which we will discuss later.

Interaction network analysis of SARS-CoV and SARS-CoV-2 spike protein complexes
The interactions pattern between the spike glycoprotein and ACE2 has been evaluated in SARS-CoV and SARS-CoV-2 using NAPS. The structures were obtained from the initial and nal 500 frames of the simulation. Our results showed that the spike is attached to the receptor through two regions at the beginning and end of the receptor-binding motif during the simulation. Although other interactions also were occurred by the end of the simulation, it seems that these two areas play an important role in the attachment of the virus spike to its cognate receptor ( Figure 3).
Analysis of binding free energies for three forms of the spike-receptor complex The MM-PBSA method was utilized for the calculation of the binding energies of SARS-CoV, SARS-CoV-2, and the chimeric structure when bound to ACE2 ( Table 1). The lowest binding energy was related to SARS-CoV-2 with -31.5759 ±2.4425 kcal.mol -1 . According to the results, electrostatic interactions have an essential role in binding a nities between SARS-CoV-2 and its receptor.
The result showed that the mutations in receptor-binding motif have an essential role in the increased a nity of SARS-CoV-2 to ACE2; however, the impact of mutations on the other regions of RBD is not great as much as mutations effects on RBM. According to the above results, interaction mechanism of SARS-CoV and SARS-CoV-2 spike with ACE2 receptor has been investigated in details and roles of mutations in changing the SARS-Co-2 a nity for ACE2 have been also assessed.

Binding free energy decomposition for spike-receptor complexes
The analysis of free energy decomposition was performed on the spike-ACE2 complexes. The results are depicted in gure 4 and tables S2 and S3. Free energy decomposition analysis helps to nd contribution of a single residues by summing its interactions over the entire residues.
Mutations Pro462Ala and Leu472Phe in SARS-CoV-2 altered the binding free energy from -2 kcal·mol −1 in SARS-CoV to -6 kcal·mol −1 . These two residues are located at the beginning and end of a loop that interacts with the N-terminal domain of the receptor. The exibility of this loop might facilitate the binding of the spike protein to its receptor which is shown in gure S6. The Pro462Ala mutation makes the region exible as a hinge, and therefore, facilitates the binding of the virus to its receptor.
Mutation Asn479Gln also altered the binding free energy of these from -2 kcal·mol −1 in SARS-CoV to -4 kcal·mol −1 in SARS-CoV-2. Therefore, it seems that this region also plays a critical role in the interaction of coronavirus to ACE2. This nding was also con rmed by the native contact result.
Also, binding free energy decompositions for ACE2 residues have been calculated, and the results are
The maximum number of native contacts in the interaction between the receptor-binding motif of the spike protein and ACE2 receptor was observed in residues 31, 353 of ACE2 for both structures (SARS-CoV and SARS-CoV-2). These two residues are considered as hotspot points that interact with the beginning and terminal regions of the receptor-binding motif in the spike protein.
In addition to regions at the beginning and terminal of the receptor-binding motif, another region in the middle of the receptor-binding motif was also involved in the interaction between the spike protein and ACE2 receptor. This region acts as a clamp in the binding of the virus to ACE2 (Fig. 6A3, 6B3). Indeed, in this region is including Tyr440, Tyr442, Leu443, and Asn479 of SARS-CoV as well as Tyr453, Phe455, Leu456 and Gln493 of SARS-CoV-2, create a cavity that, through its two edges interacts with Thr27, Asp30, Lys31, His34 and Glu35 of ACE2.
There are three mutations in this region, including Tyr442Lue, Leu443Phe, and Asn479Gln.
These mutations cause an increase in the binding a nity of the ACE2 receptor to SARS-CoV-2 compared with SARS-CoV. For more con dence, selected alanine scanning was done for residues (Tyr440, Tyr442, Leu443, and Asn479 for SARS-CoV and Tyr453, Leu455, Phe456, Gln493 for SARS-CoV-2) and MM-PBSA method was used in order to calculate the binding a nity of each substitution (Table 2 and 3). According to the result, altering each of these residues lead to reduce the binding a nity in SARS-CoV-2 and the lowest binding a nity was observed for Phe456Ala substitution.
In contrast to the selected SARS-CoV-2 alanine scanning results, except for Leu443Ala, the rest of substitution for SARS-CoV have increased the binding a nity to ACE2.

Discussion
Virus-receptor recognition is a primary viral infection phase and plays a decisive role in tissue tropism in host cells. The improved binding a nity of SARS-CoV for ACE2 has been correlated with the disease severity and virus transmissibility in humans 13,14,15,16 . The epidemiological studies indicated that the infectivity of SARS-CoV isolated from three epidemics are between 2002 and 2003 was higher and more pathogenic in humans than the isolates of the re-emergence era between 2003 2004. According to some reports, speci c mutations in the spike glycoprotein may in uence the binding a nity of SARS-CoV to ACE2 17,18,15 . In some studies, it was reported that SARS-CoV-2 employs ACE2 as an entry receptor. It was also suggested that the SARS-CoV-2-ACE2 complex has the same a nity as the SARS-CoV-ACE2 complex isolated during 2002-2003 19 . Another study reported that the binding a nity between ACE2 and the RBDs of SARS-CoV-2 and SARS-CoV has similar ranges 20, 21 .This indicates that it could be generalized to humans, as many types of SARS-CoV-2 could be transmitted from human-to-human. On the other hand, some studies reported that the binding a nity of the S-protein to the ACE2 receptor is 20 folds higher than that of SARS-CoV, as con rmed by Cryo-EM analysis of the spike protein structure in the perfusion conformation 22 .
In this study, we employed in silico methods to investigate the interaction of the spike protein of SARS-CoV and SARS-CoV-2 with the ACE2 receptor in atomic details to understand the biological process by which the virus infects the host cells. Therefore, by analysis of the detailed interaction patterns, we will able to discover new methods to neutralize virus infection. Herein, the spike-receptor interaction analyses were performed to nd hotspot residues involved in such feasible interactions. According to proteinprotein interaction results, residues 24-38 Pycontact was applied to explore the interface regions between ACE2 and spike during the simulation.
According to our results, N-terminal domain which includes residues 35-54 and 300-330 of ACE2 in regions  and 266-344, showed the highest interface area in SARS-CoV-2 and SARS-CoV complexes; however, the interface area between the SARS-CoV-2 and ACE2 in regions  increases during the simulation and also was higher than of SARS-CoV. The results also demonstrated an interface region including residues 346-372 between SARS-CoV-2 and ACE2 while no such interface region was observed between SARS-CoV and ACE2. This may be due to the fewer interactions in this area in the SARS-CoV-ACE2 complex.
After investigating the binding regions on ACE2, we focused on the H-bond patterns to understand the detailed a nity between the spike glycoprotein and ACE2 receptor in four areas separately (19-33, 35-54, 353-358 These results are probably due to mutations in the receptor-binding motif of SARS-CoV-2, resulting in an increase in the a nity to ACE2 when compared with SARS-CoV. Of note, mutations in other regions of RBD in uence the interaction of SARS-CoV-2 with ACE2 but not as much as mutations occurring in RBM.
For more details, the free energy decomposition for all residues in binding regions of the spike protein and receptor has been analyzed. We observed several mutations in the receptor-binding motif of the spike protein, leading to a change in binding free energy in the SARS-CoV-2-ACE2 complex when compared with SARS-CoV-ACE2.
Mutation Thr487Asn could explain the increment of the H-bond in region 334-339 in SARS-CoV-2 compared with SARS-CoV. Thr487 has an essential role in recognition of the human ACE2 by the SARS-CoV 26 . Ortega and colleagues also reported that mutation Thr487Asn may result in increasing binding a nity of SARS-CoV-2 with its cognate receptor 27 . Mutations Thr487Asn could also be considered as a reason for increasing of the interaction number as well as interface area in region 327-353 of ACE2 when interacting with SARS-CoV-2 compared with SARS-CoV. Other mutations including Pro462Ala and Leu472Phe are located almost at the beginning and end of the loop region (residues 475-486). Also, mutation Asn479Gln located in the middle part of the receptor-binding motif of the spike protein, leading to a decrease in the binding free energy at this point in SARS-CoV-2 compared with SARS-CoV.
These mutations (Tyr442Leu, Leu443Phe, Pro462Ala, Leu472Phe, Asn479Gln and Thr487Asn) interact with Lys31 and Lys353 of ACE2 that were previously introduced as hotspot points in the spike-ACE2 interaction 12,28 . The analysis of free energy decomposition of ACE2 indicated that the binding free energies of these residues (Lys31 and Lys353) were decreased from SARS-CoV to SARS-CoV-2. The decrease in free binding energy stems from the presence of four mutations that cause an increase in the a nity of SARS-CoV-2 to ACE2. Therefore, mutations Thr487Asn, Pro462Ala, Leu472Phe, and Asn479Gln could be considered as hotspot points in RBD of SARS-CoV2 and play an important role in the interaction of the virus with the two ends of the N-terminal domains of ACE2, leading to higher a nity of SARS-CoV-2 spike protein to its receptor compared with SARS-CoV.
In last step, native contact analysis was used to discover the more details from protein-protein interactions between spike glycoproteins and ACE2 receptor. Based on the native contact pattern results, there are three regions in the receptor-binding motif which involve in spike-ACE2 interaction, including the beginning and end of the receptor-binding motif, and a region located in the middle area of the receptorbinding motif of the spike protein in SARS-CoV and SARS-CoV-2. The middle area is comprised of two beta-sheets that form a clamp-like structure. It is now known that β-sheets play a critical role in function of proteins, such as ligand binding or target recognition domains 29 , protein-protein interactions (PPIs) 30 , and targets of proteases 31 . According to native contact results, it seems that this region plays a fundamental role in the interaction of coronaviruses with ACE2. Also, in previous studies some of the residues which are located in this area were introduced as important point for virus-spike interaction 27 .
To further con rm the importance this area, we employed selected alanine scanning for some critical residues in this region. Based on these results, altering key residues to alanine in this region lead to an increase of binding free energy in SARS-CoV-2 while the binding free energy has decease for SARS-CoV.
Finally, our results might be helpful for addressing how mutations in the receptor-binding motif play signi cant roles in increasing the a nity of the virus to its receptor, or other mutations in other points which in uence the viral infection. Protein-protein interactions (PPIs) are regularly mediated by distinct PPI domains and could be determined by analyzing the 3D structure of the domains due to the intermolecular interactions between the proteins 32 . While direct mutation in PPI area are important, other mutations in residues located outside of the receptor-binding motif could affect the 3D structure of domains and could consequently change the viral a nity to speci c receptors which has been explained for the case of chimer-ACE2 complex.

Conclusion
In the current study, molecular dynamics simulation and binding details of the spike glycoprotein-ACE2 complex for SARS-CoV, SARS-CoV-2, and chimeric structure have been performed. The present study aimed to understand the differences in the binding mechanism of the virus to its receptor in three complex forms since these structures could be regarded as a target for drug design. Also, there are mutations in speci c regions of the receptor-binding motif that may have an important effect on the spike a nity to its speci c receptor. Therefore, our ndings showed that receptor-binding motif of the protein have an essential role in the interaction of the spike protein with ACE2. Mutations in the receptor-binding motif could be regarded as hotspot points for drug design and the inhibition of the spike-ACE2 interaction.

Analysis of intermolecular interactions between the Spike (S) glycoprotein and ACE2 receptor
The structural analysis of coronavirus spike receptor-binding domain in complex with its receptor was carried out on three PDB les (2ajf, 6m0j, and 6vw1) by Swiss-PDB viewer and PDBsum LigPlot+ to assess the interaction of the protein S with its cognate receptor 35,36 .

Molecular dynamics simulation
Molecular dynamics simulation of complex structures was performed to understand the interaction between the spike glycoprotein and its receptor using AMBER20 37 and pmemd.cuda GPU code and ff14SB force-eld 38 . The complex was neutralized by adding Clions to the structure using the LEaP module. Afterward, the structures were immersed in an octahedral box lled with a 10 Å layer of TIP3P water molecules 38 . Then, the topology and the coordination were saved for the subsequent steps in simulations. The energy minimizing of the solvated spike-ACE2 complex was performed in two phases. First, the ions and water were minimized by 3000 steps; then, the entire system was minimized by 5,000 steps employing the steepest-decent and conjugate gradient algorithms. For calculating non-covalent interactions by the PME, the cutoff distance was adjusted to 10 Å in the periodic boundary condition. The system was heated from 0 to 300 K for 200 ps, with the NVT ensemble using Langevin thermostat with a collision frequency of 2 ps 39 . The bonds were constrained, including hydrogen atoms using the SHAKE algorithm 40 . Prior to the MD production, the equilibration was performed in the NPT ensemble for 1 ns with Berendsen barostat and relaxation time 2 ps, and the pressure was adjusted to 1 atm. Ultimately, MD simulation was performed for 100 ns with the NPT ensemble. The time-step was set at 2 fs, and the coordinates were saved every 0.8 ps.
Trajectory analysis: The trajectory analysis was carried out using CPPTRAJ 41 from AmberTools 20 for calculating the uctuation and native contacts.

Interaction Analysis of complexes during simulations
PyContact was used as a tool for the analysis of non-covalent interactions from trajectories. PSF (topology) and CDC (trajectory) are used as input formats then the results were expressed as contact score (the number of hydrogen bonds) and solvent accessible surface areas (SASAs) as shown by histograms or contact maps 42 .

Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculation
In order to compare the binding a nity of the spike glycoprotein-ACE2 complex for three structures (2ajf, 6m0j, and 6vw1), MM-PBSA calculation was conducted with the previous procedures using Amber 20 for 20 ns using ff99SB force-eld. The analysis of binding free energies was performed by mmpbsa.py 43 .
The network analysis of protein structures NAPS server (http://bioinf.iiit.ac.in/NAPS/) was applied to perform the network analysis of protein structures at different snapshots during the simulation process 44 .    Figure 1 Two-dimensional interaction schemes for the spike-ACE2 complex: SARS-CoV (A), chimeric structure (B), and SARS-CoV2 (C). ACE2 and spike proteins residues denoted by A and E in parenthesis respectively. Also, hydrogen bonds and hydrophobic interactions are colored in yellow and green lines respectively.  The interaction network analysis of ACE2-spike protein complex during the simulation: (A) in SARS-CoV (2ajf) and (B) SARS-CoV2 (6m0j) structures. The PDB structures obtained from the initial (A1 and A2 for SARS-CoV, and B1 and B2 for SARS-CoV-2) and nal (A2 and A3 for SARS-CoV, and B3 and B4 for SARS-CoV-2) 500 frames of the simulation were analyzed by NAPS. The red points in A1, A3, B1 and B3 indicate critical amino acids that participated in the spike-ACE2 interaction.