The recent outbreak of coronavirus disease 2019 , caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected all walks of life. Genomic studies have established that SARS-CoV-2 belong to the betacoronavirus genus, which also includes SARS-CoV and MERS-CoV that were associated with previous outbreaks of relatively smaller scale [1] [2] [3] . These coronaviruses attach to the host cell with the aid of the spike glycoprotein present on its envelope. Coronavirus spike glycoprotein is composed of two subunits -the S1 subunit is important for binding to the host cell receptor and the S2 subunit is responsible for the fusion of the virus and the host cell's membrane. Angiotensin converting enzyme 2 (ACE2), an enzyme located on the outer surface of a wide variety of cells, is the primary host cell target with which the spike protein of SARS-CoV and SARS-CoV-2 associates [4] [5] [6] . The receptor binding domain (RBD) of the S1 subunit of these viruses binds to outer surface of the claw like structure of ACE2 7 .
The sequence similarity of the RBD region of SARS-CoV and SARS-CoV-2 spike protein is between 73% to 76% 7 . Fourteen residues of the SARS-CoV spike protein RBD have been reported to interact with human ACE2. These are Tyr436, Tyr440, Tyr442, Leu443, Leu472, Asn473, Tyr475, Asn479, Gly482, Tyr484, Thr486, Thr487, Gly488, and Tyr491 8, 9 . Only eight of these residues are conserved in SARS-CoV-2 9 The equivalent conserved residues in SARS-CoV-2 are Tyr449, Tyr453, Asn487, Tyr489, Gly496, Thr500, Gly502, and Tyr491, while Leu455, Phe456, Phe486, Gln493, Gln498, and Asn501 are substituted.
The atomic level three-dimensional structure of several SARS-CoV-2 proteins have now been determined [10] [11] [12] [13] . These X-ray crystallography based structures provide remarkable insights into macromolecular structure and intermolecular interactions. However, molecular recognition and binding are dynamic processes. Molecular dynamics (MD) simulation often complement traditional structural studies for looking at the dynamics of these processes at the atomic level 14, 15 .
Such simulations can provide insights into the structural stability of macromolecular complexes, flexibility of interacting subunits and the interactions of residues in the interface.
Here, we report the stability and binding dynamics of SARS-CoV-2 spike RBD bound to ACE2, and compare this with the dynamics of SARS-CoV spike RBD, by running multiple 500 ns allatom MD simulations. High resolution X-ray crystal structures of SARS-CoV-2 spike RBD bound to ACE2 illustrate fourteen positions that are associated with the interaction between SARS-CoV/SARS-CoV-2 and ACE2. The primary objective of this study was to identify both similarities and dissimilarities in the dynamics of the interactions between SARS-CoV/SARS-CoV-2 and ACE2 and to identify key residues that could be vital to the integrity of this interface. This would provide insights into residues that could be targeted for disrupting this interface.
MD simulations of SARS-CoV-2-ACE2 and SARS-CoV-ACE2 complexes 500 ns MD simulations of SARS-CoV-ACE2 complex (PDB ID: 6M0J) and SARS-CoV-ACE2 complex (PDB: 2AJF) were performed in triplicate. One of the 500 ns simulations of SARS-CoV-2-ACE2 complex was further extended to 1 µs to ensure that the interactions are faithfully retained for a longer duration. Additionally, triplicate 500 ns simulations of a second SARS-CoV-2-ACE2 structure (PDB ID: 6LZG) was also performed to ensure that results were not biased by a single structure. The overall structural integrity of all stimulations of SARS-CoV2-ACE2 complex were retained with a Cα root mean square deviation (RMSD) from the starting structure that was less than 4 Å. In the case of the SARS-CoV-ACE2 simulations, one of simulations had a Cα RMSD that was under at 5 Å. The oscillating RMSD of this simulation was characteristic of the closing/opening motion of the ACE2 claw like structure 16 (Figures 1A and Figure 1B ). The other two SARS-CoV-ACE2 simulations had a higher RMSD that was associated with the detachment of the spike protein from one end of interface ( Figure 1B) . Interestingly, the other end remained attached as evident from specific contacts that were retained throughout the simulations.
Protein secondary structure composition and compactness, as indicated by the radius of gyration, of ACE2 and spike protein structures were also preserved throughout the simulations (Supplementary Figure 1) . This included salt bridges, hydrogen bonds, π-π and cation-π interactions (Supplementary Table   1 ). Some of these interactions were more persistent than others. The residues of SARS-CoV-2/SARS-CoV spike RBD that interacted with ACE2 consistently are shown in Figures 3A and 3B .
interfaces and the dynamics of the salient ones along the length of the simulation trajectories are shown in Figure 4 and Supplementary Figures 4 and 5 . Table 1 ). The likelihood of an interaction between Gln493 of SARS-CoV-2 and Lys31 residue of ACE2 was reported recently 7 .
Next, the backbone of Gly488 of SARS-CoV consistently interacted with Lys353 of ACE2 in all three simulations, while the Gly502 at the equivalent location in SARS-CoV-2 formed sustained interactions with Lys353 of ACE2 in only one simulation (Figures 4A and 4B) . Additionally, the side chain of Gln498 and backbone of Gly496 of SARS-CoV-2 spike RBD formed sustained interactions in three and two simulations, respectively, with Lys353 of ACE2. Such an interaction was absent in equivalent residues of SARS-CoV ( Figures 4A and 4B ). This suggests that SARS-CoV-2 and SARS-CoV differs in how they target the basic Lys31 and Lys353 residues of ACE2.
Tyr449 and Tyr489, two conserved residues in SARS-CoV-2 spike RBD, consistently interacted with Asp38 and Gln24 of ACE2 in multiple simulations. Tyr453 and Tyr505 were observed to interact with His34 and Gln37, respectively, of ACE2 for at least 80% of one simulation each ( Figures 4A and 4B) . The corresponding residues in SARS-CoV did not appear to form such sustained interactions except Tyr491 which consistently interacted with Gln37 in two simulations.
Furthermore, Gln493 and Gln498 of SARS-CoV-2 showed sustained interactions with Glu35 and Asp38, respectively, while the corresponding Asn479 and Tyr484 in SARS-CoV exhibited extremely weak interactions ( Figures 4A and 4B ). This is in agreement with recent work that shows the likely existence of Tyr449-Asp38, Tyr453-His34, Tyr489-Gln24, and Gln493-Glu35 interactions between SARS-CoV-2 spike RBD and ACE2 [16] [17] [18] .
Three residues which are mutated in SARS-CoV-2 (Ala475, Lys417 and Gly446) interacted with ACE2 residues (Gln24, Asp30 and Gln42). Such interactions were not observed in corresponding residues of SARS-CoV ( Figures 4A and 4B, Supplementary Table 1 ). Significantly, a very strong salt bridge was established and sustained between Lys417 of SARS-CoV-2 spike RBD and Asp30 of ACE2 for nearly the full duration of all simulations. Notably, this salt bridge is absent in SARS-CoV since the equivalent residue is Val404, which is incapable of forming such an interaction ( Figures 4A and 4B ). Gly446 maintained an interaction with Gln42 of ACE2 for majority of only one simulation ( Figure 4A ), while Ala475 exhibited only weak interactions with Gln24 in the simulations.
To look at the dynamics of the interface, interactions that were maintained for at least 50% of the total simulation time in three simulations in the two complexes were evaluated. Four interfacial residues in SARS-CoV-2 (Lys417, Gln493, Tyr449 and Gln498) and two in SARS-CoV (Thr486 and Gly488) maintained such interactions with four (Asp30, Glu35, Asp38 and Lys353) and two (Lys353 and Asp355) residues of ACE2 respectively (Figures 4A and 4B) . Hence, there are noteworthy differences between how the two viral spike proteins interact with ACE2 and the larger number of sustained interactions in SARS-CoV-2 spike protein could be associated with a higher binding affinity of SARS-CoV-2. Additionally, similar dynamics of interacting residues were also observed in triplicate 500 ns simulations of another complex structure of SARS-CoV-2-ACE2 (PDB: 6LZG) that was simulated to ensure that the results were not biased by one structure Recent studies, based on models of the SARS-CoV-2 spike protein RBD, have indicated that Leu455, Phe486, Ser494, and Asn501 of SARS-CoV-2 are important for binding to ACE2 via their interaction with Met82, Tyr83, Lys31, and Tyr41 residues 3,7,18 . However, from the simulations, only a weak π-π interaction was observed between Phe486 and Tyr83 (Supplementary Table 1 ). While, Leu455, Ser494, and Asn501 were not observed to form any significant interactions with ACE2.
Water molecules play an important role in many intermolecular interfaces. In this instance, six conserved water sites were found in the interface between ACE2 and SARS-CoV-2 spike RBD.
Water-mediated indirect interactions were formed between ACE2:Lys31 and spike:Phe490/Leu492, ACE2:Asp38 and spike:Gly496, ACE2:Asn33/His34/Glu37/Asp38 and spike:Arg403 ( Figure 5 ). These could also play a role in stabilizing the interface. 
This study provides insight into the stability of the interactions that define the SARS-CoV-ACE2 and SARS-CoV-2-ACE2 interfaces, using extended MD simulations of X-ray crystal structures of these complexes. Firstly, interactions that were shared by SARS-CoV-2-ACE2 and SARS-CoV-ACE2 complexes were assessed. SARS-CoV-2 spike protein RBD consistently interacted with ACE2 in three clusters. At one end, Gln493 and Gln498 formed sustained hydrogen bonds with Glu35 and Lys353 of ACE2. At the other end, Tyr449 formed hydrogen bond with Asp38 of ACE2. In the middle, Lys417 formed a strong and stable salt bridge with Asp30 of ACE2.
Additionally, several intermittent interactions of SARS-CoV-2 could permit it to bind more stably than SARS-CoV (Figures 3B and 4A and Supplementary Table 1 ). These findings were also supported by the results from two different structures of the SARS-CoV-2-ACE2 complex (Supplementary Figure 6 and Supplementary Table 1 ). Loop regions (residues 484-505) of SARS-CoV-2 fluctuated less when bound to ACE2, when compared to SARS-CoV. This is stabilized by the formation of sustained interactions between Gln493 and Gln498 of SARS-CoV-2 and Glu35 and Lys353 of ACE2, respectively ( Figures 4A and Supplementary Figure 4) .
Unlike SARS-CoV-2, in SARS-CoV, the region in the middle was devoid of any stable interactions with ACE2. However, at the two ends, a different set of residues in SARS-CoV formed interactions with ACE2 ( Figure 3D ). Therefore, it is apparent that there are several similarities and differences in the structure and dynamics of the interactions of SARS-CoV-2 and SARS-CoV with ACE2. Hence, antibodies or antiviral treatment modalities that target the spike protein of SARS-CoV is not expected to produce a similar effect with SARS-CoV-2. Some of the recent studies that failed to inhibit the binding of SARS-CoV-2 RBD to ACE2, support these findings 13, 19 .
Secondly, two charged virus binding hot spots on human ACE2 (Lys31 and Lys353), which are essential for the binding of SARS-CoV, have been studied extensively 4, 8 . Charge neutralization of these hot spot lysines has been shown to be important for the binding of coronavirus to ACE2 7, 8 . SARS-CoV-2 and SARS-CoV utilizes unique strategies to achieve this. Interestingly, SARS-CoV-2 residues only formed sustained interactions with Lys353 of ACE2 and these were absent in SARS-CoV indicating an adaptation to a stronger interface ( Figure 4A Figure   2B and 2D). The strength of this sustained salt bridge between Lys417 and Asp30 could contribute to the substantially different binding affinity of SARS-CoV-2 to ACE2 receptor when compared to SARS-CoV 16, 18 . Notably, some of the previously reported residues (Leu455, Phe456, Tyr473, Phe486, Ser494, and Asn501) that were suggested to enhance the binding affinity of SARS-CoV-2, were not observed in these simulations 3, 16, 18 .
In conclusion, while SARS-CoV-2 and SARS-CoV spike RBD bind to the same region of ACE2 and share several similarities in how they interact with ACE2, there are a number of differences in the dynamics of the interactions. One salient difference is the presence of a stable salt bridge between Lys417 of SARS-CoV-2 spike protein and Asp30 of ACE2 as well as three stable hydrogen bonds between Tyr449, Gln493, and Gln498 of SARS-CoV-2 and Asp38, Glu35, and Lys353 of ACE2, which were not observed in the SARS-CoV-ACE2 interface. Stable viral binding with the host receptor is crucial for virus entry. Thus, special consideration should be given to these stable interactions while designing potential drugs and treatment modalities to target or disrupt this interface.
Coordinates of the three dimensional X-ray crystal structures of the SARS-CoV and SARS-CoV-2 RBD in complex with ACE2 were obtained from the Protein Data Bank (PDB). The PDB IDs of the structure used are 6M0J and 6LZG for SARS-CoV-2 spike protein RBD bound to ACE2 and 2AJF for the SARS-CoV spike protein RBD bound to ACE2. Schrödinger Maestro 2019-4 (Schrödinger, LLC, New York, NY) was used to visualize and prepare the protein structures for simulations. The structures were first pre-processed using the Protein Preparation Wizard (Schrödinger, LLC, New York, NY). The protein preparation stage included proper assignment of bond order, adjustment of ionization states, orientation of disorientated groups, creation of disulfide bonds, removal of unwanted water molecules, metal and co-factors, capping of the termini, assignment of partial charges, and addition of missing atoms and side chains. In the case of the SARS-CoV structure, a loop (residues 376-381) missing in the PDB structure was modeled using Schrödinger Prime 20 . Hydrogen atoms were incorporated, and standard protonation state at pH 7 was used. Structures of spike protein RBD bound to ACE2 were placed in orthorhombic boxes of size 125 Å × 125 Å × 125 Å and solvated with single point charge (SPC) water molecules using the Desmond System Builder (Schrödinger, LLC, New York, NY). A box size of 85 Å × 85 Å × 85 Å was used for simulations of spike RBD structures isolated from the PDB structures 6M0J and 2AJF. Simulation systems were neutralized with counterions and a salt concentration of 0.15M NaCl was maintained. MD simulations were performed using Desmond 21 . The OPLS forcefield was used for all calculations. All systems were subjected to Desmond's default eight stage relaxation protocol before the start of the production run. 500 ns simulations were performed in triplicate with a different set of initial velocities for simulations involving 6M0J, 6LZG and 2AJF while one 500 ns simulation each were run for the isolated spike protein structures. One of the 500 ns simulations of the SARS-CoV-2-ACE2 complex was extended to 1 µs to ensure that the interactions are retained for a longer period. For the simulations, the isotropic Martyna-Tobias-Klein barostat and the Nose-Hoover thermostat were used to maintain the pressure at 1 atm and temperature at 300 K, respectively 22,23 . Short-range cutoff was set as 9.0 Å and long-range coulombic interactions were evaluated using the smooth particle mesh Ewald method (PME) 24 . A time-reversible reference system propagator algorithm (RESPA) integrator was employed with an inner time step of 2.0 fs and an outer time step 6.0 fs 25 . Simulation data was analyzed using packaged and in house scripts and plotted using R 3.6.0 (https://www.r-project.org).