Structural and simulation analysis of hotspot residues interactions of SARS-CoV 2 with human ACE2 receptor

Abstract The novel corona virus disease 2019 (SARS-CoV 2) pandemic outbreak was alarming. The binding of SARS-CoV (CoV) spike protein (S-Protein) Receptor Binding Domain (RBD) to Angiotensin converting enzyme 2 (ACE2) receptor initiates the entry of corona virus into the host cells leading to the infection. However, considering the mutations reported in the SARS-CoV 2 (nCoV), the structural changes and the binding interactions of the S-protein RBD of nCoV were not clear. The present study was designed to elucidate the structural changes, hot spot binding residues and their interactions between the nCoV S-protein RBD and ACE2 receptor through computational approaches. Based on the sequence alignment, a total of 58 residues were found mutated in nCoV S-protein RBD. These mutations led to the structural changes in the nCoV S-protein RBD 3d structure with 4 helices, 10 sheets and intermittent loops. The nCoV RBD was found binding to ACE2 receptor with 11 hydrogen bonds and 1 salt bridge. The major hot spot amino acids involved in the binding identified by interaction analysis after simulations includes Glu 35, Tyr 83, Asp 38, Lys 31, Glu 37, His 34 amino acid residues of ACE2 receptor and Gln 493, Gln 498, Asn 487, Tyr 505 and Lys 417 residues in nCoV S-protein RBD. Based on the hydrogen bonding, RMSD and RMSF, total and potential energies, the nCoV was found binding to ACE2 receptor with higher stability and rigidity. Concluding, the hotspots information will be useful in designing blockers for the nCoV spike protein RBD. Communicated by Ramaswamy H. Sarma


Introduction
The emergence of highly pathogenic novel coronavirus in 2019 (SARS-CoV 2) became pandemic and posed a global health emergency. The Coronaviridae family viruses are mostly hosted by animals and are found pathogenic to humans. Though these viruses were pathogenic to humans, most of them caused mild to moderate symptoms (Lu et al., 2020). However, Severe Acute Respiratory Syndrome (SARS) in 2002 and the MERS (Middle East Respiratory Syndrome) in 2012 caused by SARS-CoV (CoV), infected larger populations and showed significant fatalities. In December 2019, another beta-Coronavirus has been identified in a group of people admitted with severe pneumonia symptoms in Wuhan, China named as SARS-CoV 2 (nCoV) (Lu et al., 2020;Muralidharan et al., 2020;Peeri et al., 2020;Umesh et al., 2020;Wahedi et al., 2020;. In a short span of time, the infection rate increased with $4.2 million people infected and 0.29 million deaths by 14 th May 2020 throughout the world (WHO, 2020) and continuing. Many of the countries adopted social distancing and lock down precautions to decrease the rate of nCoVID-19 infections from human to human but for the lack of vaccines and targeted therapies (Aanouz et al., 2020;Elfiky, 2020a;Kumar et al., 2020).
The recently published crystal structure (PDB Id: 6M0J) elucidates the structural interactions of spike protein RBD of nCoV. Considering that there are extended mutations present in the nCoV sequence, which comes under RBD (Sinha et al., 2020), it has been shown that more ACE2 interacting residues were present in the nCoV spike protein RBD than CoV (Lan et al., 2020;Pant et al., 2020). It is important to understand the hot spot amino acids and their interactions and how far these interactions are stable allowing the membrane perfusion of virus into the host cell. The current in silico study focuses on, highlighting the ACE2 and spike protein RBD amino acid interactions. The strength of the hot spot amino acid interactions were studied through molecular dynamic simulations for a period of 100 ns. The deviations and fluctuations made by the ACE2-RBD complex along with energies clarify in understanding the stability of the nCoV spike protein RBD interaction with ACE2 receptor in comparison to SARS-CoV.

Materials and methods
The entire computational work was performed using the Schrodinger suite. The applications employed in the present study are

Multiple sequence alignment
The mapping of S protein RBD sequences of both CoV and nCoV was performed using the multiple sequence viewer tool of the prime application of Schrodinger suite.

Protein preparation wizard
The crystal structures were retrieved from protein databank and prepared using protein preparation wizard tool. The parameters used in refining the structure are addition of hydrogen's, creating disulphide bonds, maintaining zero order bonds and selenomethionines to methionines conversion in the import and process tab. Further, in refine tab, optimizing the hydrogen bonds to repair and finally minimized the structure through force field OPLS_2005. Using superimposition tool of the maestro, both complexes (CoV and nCoV-ACE2) and individual S-protein RBD of both CoV and nCoV were structurally superimposed and their RMSD (Root Mean Square Deviations) was calculated (Jorgensen et al., 1996;Sastry et al., 2013;.

Molecular dynamics simulations
Molecular dynamic simulations (MDS) of the complexes were performed using Desmond software. Initially, the complex was imported into the system builder application of Desmond module and with default parameters like SPC (simple pointcharge) solvent model, orthorhombic periodic boundary box (Box size; distances (Å): a:10 Â b:10 Â c:10 and Angles: a:900 Â b:900 Â c:900) and minimizing the volume, a model system was generated for simulations. Continuing with the ions tab of system builder application, Na þ ions were added based on the total charge and a salt concentration of 0.15 M was also added to neutralize the system. Second step in the simulations protocol was minimization, the complex obtained from the system builder was relaxed by setting the maximum iterations number to 2000 and remaining parameters were set to default. Finally, the minimized complex was subjected to molecular dynamic simulations by setting the ensemble parameter to NPT [isothermal-isobaric ensemble, Number of particles (N), Pressure (P) and Temperature (T)], 300 K temperature, 1 bar pressure, simulation run time was set to 100 ns (Islam et al., 2020) and relaxed using the default relation protocol (Guo et al., 2010;Veeramachaneni et al., 2019).

Protein binding analysis
Protein binding analysis was performed using the protein interaction analysis application of the Schrodinger suite.

Sequence analysis of SARS-Corona virus and SARS-Corona virus-2 spike protein
Spike protein (S-protein) sequence of SARS-CoV (CoV) and SARS-CoV 2 (nCoV) were retrieved from the uniprot databank with sequence IDs P59594 and P0DTC2 respectively. The multiple sequence alignment was performed using Clustal omega web server of EMBL-EBI services. The alignment results displayed 75.9% identity ( Figure 1) between the sequences. Previous studies (Aydin et al., 2014) reported the Receptor Binding Domain (RBD) of the CoV spike protein ranging from 306 to 527 and in specific the residues 424-494 related to binding motif played a crucial role in binding to the human ACE2 receptor for viral admittance. In nCoV, these residues were aligned at 319-541 and 437-508 positions respectively. The spike protein RBD of CoV and nCoV shared only 74% identity. The alignment displayed 26% mutations and 58 residues were found mutated in the spike protein RBD region of nCoV. Among these, 34 mutations related to the binding motif were predicted as the crucial amino acids based on the correlation analysis done with the spike protein binding motif of CoV. Considering this binding region as a major criterion, the CoV and nCoV receptor binding domains were further analyzed for any structural changes resulting out of these mutations.

Structural analysis of CoV and nCoV receptor binding domain
To continue the structural analysis of the receptor binding regions, reported PDB structures of SARS-CoV (PDB Id: 3SCI) and SARS-nCoV (PDB Id: 6M0J) were retrieved from the protein databank. The F-chain in both the sequences relates to the spike protein RBD of the virus and the A-chain represents ACE2 receptor. Prior to the analysis, RBD sequences retrieved (PDB) were cross validated against their corresponding full length uniprot sequences. Many structural modifications were observed in the nCoV RBD of Spike protein because of the mutations occurred during the course of time compared to the CoV. The overview of the mutational changes in the secondary structure of both CoV and nCoV spike protein receptor binding region were aligned in Figure  2(a). In CoV, the majority of the spike protein RBD 3d structure was made of loops with 2 helices and 5 sheets. While, the 3d structure of nCoV spike protein RBD comprised of 4 helices and 10 sheets with interconnecting loops ( Figure  2(b)) (Lan et al., 2020). The detailed information regarding the residues involved in the helices and sheets of both the proteins were tabulated ( Table 1). The major structural changes recorded were seen in the residues ranging from 306 to 382 of CoV which were in the loop region while the corresponding residues of nCoV i.e. 333-395 were framed into two helices and three sheets. The helix observed in the CoV protein (426-429) was absent in the corresponding nCoV (439-442 residues) and these residues were sited in loop region. Extra sheets in nCoV protein between Tyr 473-Gln 474, Cys 488-Tyr 489 and Val 524-Cys 525 were absent in CoV and these residues were shaped into loops.

Binding modes of CoV and nCoV spike protein receptor binding domain with ACE2 receptor
The complex of the ACE2 receptor with RBD of the CoV and nCoV spike protein crystal structures were retrieved and prepared using the protein preparation wizard application of the Schrodinger suite through a series of steps as explained in the methodology part. The prepared complexes were subjected to protein interaction analysis application available in the suite to investigate the interactions occurring between the receptor and spike protein RBD. The superimposition of CoV and nCoV without ACE2 receptor resulted in 7.37 Å RMSD (Figure 2(c)) and in complex with ACE2 receptor, the superimposed complexes were displaying 15.24 Å deviations (RMSD) (Figure 3(a)). The CoV RBD-ACE2 complex was maintained with 7 Hydrogen bonds, 1 pi-pi interaction and 1 Salt bridge ( Figure   3(b)). Concurrently, the nCoV spike protein RBD-ACE2 receptor complex displayed 11 hydrogen bonds and 1 salt bridge (Figure 3(c)). Interactions like hydrogen bond, pi-pi stacking and salt bridge made by the CoV and nCoV spike protein with ACE2 receptor were reported along with their closest residues, distance and also the solvent accessible surface area (SASA) ( Table 2). In both the complexes, residues 473/ 487, 436/449 and 473/487 of virus RBD maintained hydrogen bond interaction profiles with the residues 24, 38 and 83 of the ACE2 receptor respectively. Prime/MM-GBSA based binding-free energy was calculated by considering nCoV and CoV as ligands against the ACE2 receptor. The binding-free energies calculated were À126.5 kcal/mol for nCoV and À100.2 kcal/mol with CoV. Comparing the two complexes binding interaction profiles, nCoV was found binding more rigidly to the ACE2 receptor than CoV with more number of H bonds and the binding free energy calculations were also found inline supporting the interaction analysis results confirming nCoV binding with more stringency to the human ACE2 receptor when compared to CoV. . Molecular dynamic simulation analysis of ACE2-CoV S-protein RBD and ACE2-nCoV S-protein RBD a) Deviations graph of ACE2 receptor and CoV S-protein RBD. b) RMSD graphs of ACE2 receptor and nCoV S-protein RBD. c) RMSF graph of ACE2 receptor and CoV S-protein RBD. d) ACE2 receptor and nCoV S-protein RBD residues fluctuation graph.

Molecular dynamic simulations
Both the crystal structures were subjected to molecular dynamic simulation studies over a period of 100 ns to understand the stability of the complexes. Hydrogen bond interactions, Root-Mean-Square Deviation (RMSD) and Root-Mean-Square Fluctuation (RMSF) made by CoV and nCoV with the ACE2 receptor in complex state and their binding profiles were analyzed. The average total energy and potential energy of the complexes were also calculated to evaluate the rigidity of the complexes.

Deviations analysis
Using the trajectories of the complex, the deviations made by the two spike proteins were reported individually. The deviations made by the ACE2 receptor and spike protein RBD of CoV complex (3SCI) were displayed in the Figure 4(a). In the initial simulation period until 30 ns, the ACE2 receptor deviations were found in the inclined state and thereafter the protein maintained stable deviations till the end of the simulation run time (100 ns). The deviation range was between 2.0 and 4.0 Å while majority of the deviations were around 3.5 Å. The spike protein RBD inclined deviations were observed throughout the simulation run time and the deviations were in the range of 3.0-5.0 Å.
In the ACE2 -nCoV spike protein RBD complex (6M0J), the ACE2 receptor made deviations between 2.5 and 3.5 Å and majority of the deviations were maintained around 3.0 Å. From 20 ns of simulation run time, steady state in the deviations with small fluxes were observed till the end of 100 ns (Figure 4(b)). Based on the acceptable deviation range i.e. 2-3 Å, the deviations made by the ACE2 receptor reflects the stability of the receptor in the complex form. The deviations made by the nCoV spike protein RBD were low raging between 2.0 and 3.0 Å only. The stability in the deviations Figure 5. Hydrogen bond and energies reported by the ACE2-CoV S-protein RBD (3SCI) and ACE2-nCoV S-protein RBD (6M0J) after simulations. a) Hydrogen bonds graph maintained by the receptor and protein during simulation run b) ACE2 receptor and S-protein RBD of nCoV complex hydrogen bonds graph after simulations studies. c) Average energy and potential energies graph of ACE2-CoV S-protein RBD complex and d) Average energy and potential energies graph of ACE2-nCoV Sprotein RBD complex.
was attained from the 40 ns run time and maintained the pace till the end of the simulation run. Comparing the two complexes based on the RMSD results, the ACE2-nCoV was found more stable compared to the ACE2-CoV complex. The spike protein RBD of CoV and nCoV deviations difference was 2.0-2.5 Å which is higher when compared to the ACE2 receptor with a change of around 0.5-1 Å only. The receptor made deviations were almost similar in both the complexes while the difference in the deviations made by CoV and nCoV spike protein RBD could be attributed to mutations and structural changes.

Residues fluctuation analysis
Fluctuations made by the ACE2 receptor and CoV spike protein RBD were depicted in the Figure 4(c). The range of the ACE2 receptor residues fluctuation with CoV was between 0.5 and 3.0 Å while the majority of the residues fluctuated between 1 and 2Å. Interestingly, the residues of the ACE2 receptor in complex with nCoV spike protein RBD also produced a similar type of fluctuation range i.e. 0.5-3.0 Å (Figure 4(d)). CoV spike protein RBD showed fluctuations ranging 0.5-4 Å with average fluctuations around 2.0 Å while nCoV spike protein RBD fluctuations were reported between 0.5 and 2.8 Å and the majority of the residues fluctuated around 1.5 Å. The tail end residue fluctuations were also observed as expected. In conclusion, considering all the fluctuations made by the spike protein RBD in both the complexes, the nCoV residues fluctuations range was low, leading to more stability and rigidity of the complex.

Hydrogen bonds and energy
Using the trajectories and simulation event analysis, the number of hydrogen bonds formed between the complexes during the simulation runs was calculated. In ACE2-CoV spike protein RBD complex (PDB: 3SCI) the hydrogen bond range was 1-11 and mean value (average number of hydrogen bonds) was 5 ( Figure 5(a)). Similarly in ACE2-nCoV spike protein RBD complex (PDB: 6M0J) the range was 5-19 and the mean value was 11 ( Figure 5(b)). Using the simulation quality analysis tool, the total and potential energies of the two complexes were calculated. The CoV complex reported with À236309.88 and À288318.27 kcal/mol of total and potential energy whereas nCoV showed À259769.80 and À316730.74 kcal/mol respectively ( Figure 5(c, d)). These values state that the nCoV complex was more stable compared to the CoV complex justifying the increase in the average number of hydrogen bond.

Identification of crucial residues involved in hydrogen bonding: ACE2-nCoV
The main aim of this interaction profile study was to elucidate the residues (hotspots) in both the receptor and S- Table 5. List of important residues identified in ACE2 receptor and their hydrogen bond interactions sustained during the 100 ns simulation run time.
The hydrogen bonds were characterized into strong (2.2-2.5 Å), moderate (2.5-3.2 Å) and weak (3.2-4.0 Å) based on the distance between the donor and acceptor (Jeffrey & Jeffrey, 1997;Khan, Jha, et al., 2020). Based on this, majority of the reported hydrogen bonds between ACE2 receptor residues with nCoV spike protein RBD were under moderate category. The interaction percentage (100% correlating to the hydrogen bond sustainability in all the selected 10 frames) of the ACE2 receptor residues with nCoV spike protein RBD were Glu 35: 100% which preserved hydrogen bond interaction in all selected frames; Tyr 83 and Asp 38: 90%; three residues Lys 31, Glu 37 and His 34: 80%; followed by Lys 353 and Asp 30: 70%; Gln 24 and Gln 42: 50% and the remaining other five residues reported below 50% hydrogen bond sustainability (Table 5).
The corresponding nCoV amino acid residues involved in the hydrogen bonding with the receptor during the simulation studies were also identified (Table 6). Residue Gln 493 showed 100% hydrogen bonding throughout the simulation. Other residues hydrogen bond interaction percentage were Gln 498: 90%; Asn 487, Tyr 505 and Lys 417: 80%; followed by Tyr 489, Asn 501 and Tyr 453:60% and Ala 475:50%. Apart from these, the remaining other nine residues reported below 50% hydrogen bonding interaction percentage.
During simulations, some hydrogen bonds between nCoV S-protein RBD and ACE2 receptor residues were intact throughout the run time while certain residues showed shift in the hydrogen bonds. Interestingly, top listed residue Gln 493 of nCoV S-protein and Glu 35 of receptor were maintained hydrogen throughout the simulation. In addition, Gln 493 also maintained another hydrogen bond with Lys 31(70%) of the receptor and hence based on these observations Gln 493 was reported as the crucial residue in nCoV binding. Residue Gln 498 was seen maintaining an overall 90% hydrogen bond interaction shared with Asp 38 (70%) and in some intervals shifted to Lys 358 (20%).
Third residue in the nCoV S-protein RBD was Asn 487 which maintained its 90% of interaction profile with Tyr 83. Simultaneously, a shift in the H bond was also found among the residues like Lys 31 of receptor forming hydrogen bonds with Phe 490, Leu 492 and Glu 484 of the S-protein during the entire simulation period. These changes could be attributed to the flexibility nature of both receptor and S-protein during the course of the simulations run. CoV S-protein RBD residues 436, 473, 475, 479, 481, 482, 486, 487, 488 and 491 were previously reported as hotspots in several studies (Han et al., 2006;Wan et al., 2020). In the present study, comparing the hotspots of CoV and nCoV it can be stated that most of the hotspots of CoV were replicated in nCoV too. Among them, two residues Asn 479/Thr 487 of CoV were found mutated to Gln 493/Asn 501 in nCoV. These mutated animo acid residues were found having H bond interactions Gln493 (100%) and Asn 501(60%) during the simulations and profoundly influenced the binding to S-protein RBD to ACE2 receptor. The important hot spots identified in ACE2 receptor and nCoV S-protein RBD after simulation studies were displayed in the Figure 6. Along with the hydrogen interactions, hydrophobic interactions between the nCoV S-protein RBD and ACE2 receptor were also analyzed in the time frames defined (Tina et al., 2007). Based on the analysis, Leu 79, Met 82, Tyr 83, Phe 28of ACE2 receptor and Phe 486, Tyr 489 of nCoV S protein RBD were found involved in the hydrophobic interactions.

Conclusions
In conclusion, this study reports the hotspot amino acid residues involved in the binding of the nCoV spike protein RBD with human ACE2 receptor. Overall, the mutations identified in nCoV spike protein receptor binding domain had led to structural changes with additional sheets and helices in the RBD structure facilitating more H bond interactions between the ACE2 receptor and spike protein. The hotspot residues interactions were listed and the RMSD and RMSF of the 100 ns simulation run confirms the stability and rigidity of the interactions. The energy calculations reiterate the binding efficiency of the nCoV in comparison to CoV. Further, considering these hotspot amino acid residues, blockers could be designed for inhibiting the binding of nCoV spike protein RBD to human ACE2 receptor and thereby stopping the entry of the virus into the host cells.