key: cord-0895207-o8nzpx5l
authors: Giron, Carolina Corrêa; Laaksonen, Aatto; da Silva, Fernando L. Barroso
title: On the interactions of the receptor-binding domain of SARS-CoV-1 and SARS-CoV-2 spike proteins with monoclonal antibodies and the receptor ACE2
date: 2020-05-15
journal: Virus Res
DOI: 10.1016/j.virusres.2020.198021
sha: abe40a73eaa56632fd3ad7d007a3abbf937e8b7d
doc_id: 895207
cord_uid: o8nzpx5l

A new betacoronavirus named SARS-CoV-2 has emerged as a new threat to global health and economy. A promising target for both diagnosis and therapeutics treatments of the new disease named COVID-19 is the coronavirus (CoV) spike (S) glycoprotein. By constant-pH Monte Carlo simulations and the PROCEEDpKa method, we have mapped the electrostatic epitopes for four monoclonal antibodies and the angiotensin-converting enzyme 2 (ACE2) on both SARS-CoV-1 and the new SARS-CoV-2 S receptor binding domain (RBD) proteins. We also calculated free energy of interactions and shown that the S RBD proteins from both SARS viruses binds to ACE2 with similar affinities. However, the affinity between the S RBD protein from the new SARS-CoV-2 and ACE2 is higher than for any studied antibody previously found complexed with SARS-CoV-1. Based on physical chemical analysis and free energies estimates, we can shed some light on the involved molecular recognition processes, their clinical aspects, the implications for drug developments, and suggest structural modifications on the CR3022 antibody that would improve its binding affinities for SARS-CoV-2 and contribute to address the ongoing international health crisis.

The SARS-CoV-2, virus recently found in Wuhan, Hubei province, China and officially named by the World Health Organization (WHO), 1 has already spread through China from all continents (more than 168 countries), with 1,133,758 confirmed cases globally and 62,784 deaths (data as reported by Central European Time 5 April 2020). Due to the pandemic, the disease is not only affecting the health services, but also the economy in a global scale, interfering in the widespread displacement of people, tourism, local and even international markets. Once China's economy is a worldwide reference, its disruption leads to a global impact in the supply chains and the production itself. [1] [2] [3] The Coronaviridae family, to which SARS-CoV-2 belongs, includes a large variability of viruses and became recognized in the spring of 2003, when a human coronavirus caused severe acute respiratory syndrome (SARS). [4] [5] [6] [7] Based on phylogenetic analysis, the SARS-CoV-2 is classified as a lineage B betacoronavirus 8 and belongs to the same group as SARS-CoV-1 and HKU9-1, the bat coronavirus, demonstrating wide similarity with both genetically 9 (96,2% of sequence identity with HKU9-1). 10 The transmission was confirmed to be human-to-human once several medical care personnel and relatives got infected, 8, 9 but it is believed that it all started with an animal host, may it be a bat or another intermediate host. 7, 9, 11, 12 Even though the virus usually does not cause severe damage to the body, as will be explained below, the major concern is its high infectivity and pathogenicity. 8, 12, 13 The COVID-19 disease, caused by SARS-CoV-2 virus, 14 generally causes mild upper respiratory tract infections, resulting in fever and cough, yet it can also affect the lower respiratory tract. 12, 15, 16 SARS-CoV-2, on the other hand, usually remains asymptomatic in an early stage and then manifests itself with dyspnea, severe pneumonia and even death, 17 with fatality rates of about 10%. 18 Although many groups of researchers are combining their efforts to solve the mysteries of the new virus, some issues are still uncertain. Examples of these queries are the virus' incubation period, that may be longer than the 14 days scientists believed it to be previously, and the fatality rates for each age range. 19 Despite the fact that the virus' molecular mechanism is partially unknown, the SARS-CoV-2 has proteins, such as the Spike (S) glycoprotein, that densely decorates the viral external J o u r n a l P r e -p r o o f surface and can potentially be a key target for the development of vaccines and therapeutic antibodies (Abs). 8, [20] [21] [22] Due to the similarity of the receptor binding domain (RBD) in SARS-CoV-2 and SARS-CoV-1, the first strategy that has been used is to search for Abs that succeed interacting with both, once SARS-CoV-1 has been more widely studied. However, preliminary experimental studies have shown that many Abs that successfully interact with SARS-CoV-1 do not bind with SARS-CoV-2. 8 The spike protein, which is responsible for the -corona‖ (Latin word for crown) appearance in all coronaviruses, is a type I glycoprotein that has an especial role in the interaction between the virus and the host cell. This protein attaches itself to specific cellular receptors and suffers a conformational change that enables the fusion of the virus and the cell. 4, 23, 24 Studies have shown that the SARS-CoV-2's S RBD protein interacts strongly with the Angiotensin-converting enzyme 2 (ACE2). 9, 24, 25 Therefore, aiming to develop better diagnosis tools, vaccines and therapeutic Abs, it was measured the competition of mAbs and the ACE2 for the binding to SARS-CoV-2 (named before 2019-nCoV 8, 26 ) RBD protein in order to enlighten the binding epitopes of these Abs. 8, 19 The focus of this article is to initially reproduce the observations of previous laboratory experiments by a theoretical approach. Secondly, we aim to contribute with the understanding of the molecular mechanisms involved in the SARS viral infection, and finally to show how to apply this knowledge to design new functional molecules. To achieve these goals, it was tested by constant-pH simulation methods the complexation between the S RBD proteins of SARS-CoV-1 and SARS-CoV-2 with the fragments of the monoclonal Abs (mAbs) 80R, CR3022, m396 and F26G29, measuring their binding affinities and quantifying the titratable amino acids that are involved in these interactions. Thus, using a theoretical method recently proposed to identify -electrostatic epitopes‖, 27 it is possible to identify the similarities and differences between these molecular complexes, and to map their origin and possible biological implications.

Another aspect discussed in this research is the interaction between the S RBD protein from these viruses and the ACE2 in order to discover if the S RBD protein binds to either of them with higher affinity, because, if so, an antibody (Ab) might have smaller chances of binding. All this information together provided important insights to design more specific and J o u r n a l P r e -p r o o f effective neutralizing Abs which is relevant for the future prevention and treatment of this now widespread illness that should be immediately controlled. At the end, a new designed mAb candidate is proposed based on our present in silico findings.

Computational virology is an emergent research field that takes advantage of the progress from molecular and structural biology, immunology, bioinformatics and related areas to foster the understanding of virus, their evolutionary dynamics in nature, infectivity, pathogenesis, cell/hosttropism, viral assembly and their molecular interactions in general (including how to predict epitopes, how to design specific neutralizing antibodies and basically any drug design & discovery related to viral infections). [27] [28] [29] [30] [31] [32] [33] [34] In particular, structural and interactive aspects can benefit from the solid foundations that computational molecular simulation methods such as Molecular Dynamics (MD) 35, 36 and Monte Carlo (MC) 35, 37 have achieved to probe the thermodynamic, dynamics and interactive properties of biomolecules in material science, food and pharma (see refs. 38, 39 for reviews). Here, we applied a fast constant-pH MC scheme 40,41 for protein-protein studies 42, 43 to improve our understanding of the molecular interactions involving SARS-CoV-1 and 2 S RBD proteins and to identify key amino acids for the host-pathogen interactions.

Several molecular systems were investigated in the present study employing the two SARS-CoV-1 and 2 S RBD proteins (see Figure 1 ) with ACE2 and the fragments of the mAbs 80R, CR3022, m396, and F26G29. Typically, these fragments of mAbs are fusion proteins from J o u r n a l P r e -p r o o f variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. Additional calculations were carried out for the SARS-CoV-2 S RBD with a new proposed mAb based on CR3022. For most of these macromolecules, three dimensional crystallographic structures are available at the RCSB Protein Data Bank (PDB): 44 a) the SARS-CoV-1 S RBD protein was extracted from the PDB id 2AJF (chain E, resolution 2.9Å, pH 7.5)

where it was found complexed with ACE2 (chain A) − see Figure 2 ; b) the fragment of the Ab 80R was taken out from the PDB id 2GHW (chain B, resolution 2.3Å, pH 4.6); c) the anti-SARS-CoV-1 m396 Ab was extracted from the PDB id 2G75 (chains A and B, resolution 2.28Å, pH 8.5) removing part of the chains to keep only the variable regions and the short linker peptide; d) F26G19 Fab was taken out from PDB id 3BGF (chains L and C, resolution 3.0Å, pH 5.5), following the same procedure used for m396. Missing regions in these proteins were built up using the -UCSF Chimera 1.11.2‖ interface 45 of the program -Modeller‖ with default parameters. 46 Figure 3 shows the molecular structures of the SARS-CoV-2 S RBD complexed with these fragments of the mAbs. The isolated fragments of these mAbs in their final threedimensional structures as used in this work are given in Figure S2 . All PDB files were edited before the calculations. Water molecules and hetero atoms were completely removed from all used files. The -UCSF Chimera 1.11.2‖ package 45 was employed for all molecular visualizations and representations too.

When this study started, no experimental structure was available for the RBD of SARS-CoV-2 S protein. A model was built up at the SWISS-MODEL workspace (YP_009724390.1) based on the NCBI reference sequence NC_045512. 47 The root-mean-square deviation (RMSD) of atomic positions between this modeled structure for the RBD of SARS-CoV-2 S protein and the J o u r n a l P r e -p r o o f available one for SARS-CoV-1 (PDB id 2AJF) is 0.638Å . The structural comparison between the RBD proteins of both SARS viruses can be seen in Figure 1 . This field is evolving so fast that new experimental structures are continuously been solved. For example, a cryo-EM structure is now available for the prefusion S glycoprotein (a homotrimer structure) with an incomplete RBD (PDB id 6VSB, resolution 3.46Å). This study revealed an interesting pivotal movement of the RBD between two transition states (-up‖ or -down‖ conformations) with direct implications for the binding of ACE2 and/or Abs. 87 At least two monomeric S units need to be at the -up‖ conformation and properly rotated to remove the steric hindrance which allows the binding. 87, 88 This implies at least a two-step -expose-dock-like‖ mechanism where the RBD has to be exposed towards the solution in the first step (characterized by important conformational adjustments of the whole homotrimeric structure) followed by the bind or dock phase where with the targeted-RBD can interact with other molecules. Here, the focus is at this second step of this mechanism where we assume that a single targeted-RBD structure is at its final state and prepared for the binding with the ligands (i.e. at least two S monomers were already at the -up‖ conformation, correctly rotated and there was no clashes between the targeted-RBD with any other parts of the trimeric structure that could prevent the binding). 87, 88 This is the same assumption for the available crystallographic structures (e.g. PDB id 2AJF). The conformational changes between the -up‖ and -down‖ conformations (the -expose‖ phase) are not investigated in this work.

The RMSD between our model and the S RBD (chain A) from this structure is 0.790Å . This number is closer to the RMSD differences between two chains of the same experimental (PDB id 6VSB) trimer structure (e.g. 0.668Å for chain A x chain B, and 0.732Å for chain A x chain C).

Even more appropriate for the present discussion was the subsequent contribution by Yuan and co-authors that determined the crystal structure of the complex S RBD-CR3022 (PDB id 6W41, resolution 3.1Å, pH 4.6). 88 Only standard amino acids of chain A (ACE2) and E (SARS-CoV-1 S RBD) are shown in a molecular representation using spheres for its atoms. Atoms are colored accordingly to their amino acids physical chemical properties: red for acid amino acids, blue for base amino acids and gray for non-titrating amino acids. For a better visualization of the interface, the ACE2 structure was translated ~12 Å.

J o u r n a l P r e -p r o o f CR3022 is a particularly successful SARS-CoV-1 neutralizing human mAb first isolated from a convalescent patient by ter Meulen and co-authors. 48 For the present study, its three dimensional structure (see Figure 3 ) was built up at the SWISS-MODEL workspace 47 from the linear sequences of the variable regions of the heavy and light chains that were deposited in GenBank under accession numbers DQ168569 and DQ168570, respectively. 48 The RMSD between our computed generated model of CR3022 and the crystallographic structure (PDB id 6W41) is 0.537Å . This is again in the range of typical values seen for different structures determined at the same pH condition (e.g. 0.601Å for the comparison between PDB ids 6YM0 and 6YLA). Similarities with a high score are marked with -;‖ and c) the ones with low positive score are indicated by -.‖. Gaps are represented by --‖. Numbers are used to guide the identification of the amino acids sequence numbers. See text for more details.

A large diversity of models is available for MD and MC molecular simulations. 38, [49] [50] [51] [52] The need to repeat the calculations on several different physical chemical conditions and to obtain free energy of interactions at them drives the options to the so-called cost-effective coarse-grained (CG) models. These CG models offer the possibility to explore the main physical features of a system with a reduced number of parameters and lower computational costs. 27, 42, 43 During the last years, a fast constant-pH (CpH) CG model has been devised to successfully study proteinprotein interactions of several biological systems (including host-pathogens interactions). 27, 42, 43, 53, 54 The possibility to fully consider the pH effects makes this modeling approach more appealing and appropriated to address this problem. 27, 55 J o u r n a l P r e -p r o o f A sketch of the simulations model is given in Figure 5 . The S RBD proteins and the fragments of the mAbs were modeled as rigid bodies (i.e. bond lengths, angles, and dihedral angles are kept fixed) formed by a set of amino acids placed at positions given by their three-dimensional structures as described above. This additional approximation is justified by the prohibitive J o u r n a l P r e -p r o o f computational costs of constant-pH methods with pH-dependent conformational changes. 52, 56, 57 Moreover, it is known that SARS-CoV-1 S protein does not exhibit large conformational changes upon the binding to ACE2 at least. 58 Each group of atoms that define an amino acid is converted in a single charged Lennard-Jones (LJ) sphere of radius (R i ) and valence z i . This CG process turns a protein atomistic structure as a collection of charged LJ particles representing their amino acids. The centers-of-masses of the beads (mimicking amino acids) are used to place them accordingly to the coordinates given by the three-dimensional structures. The values of R i for each type of amino acids were taken from ref. 53 . The valences of all ionizable residues are a function of the solution pH. The fast proton titration scheme (FPTS) 40, 41, 52 was employed both to initially assign these valences z i´s for the amino acids and to let them vary during the simulation sampling at a given pH. This method has proved to predict pK a´s with a very good accuracy at low computational costs. 41 The fundamental physical chemical basis of this titration scheme, its numerical implementation, benchmarks, discussions related to its approximations, pros and cons can be found in previous publications. 40, 41, 52, 59 As illustrated in Figure were represented implicitly using a screening term, i.e., for two ionizable amino acids i and j, the screening is given by [exp(-κr ij )] where κ is the modified inverse Debye length, and r ij is the interparticle separation distance. 27, 42, 43, 60 Additionally, a simplified simulation box with only one protein present was used to characterize the titration properties of a single macromolecule.

The electrostatic interactions between any two ionizable amino acids of valences and are given by:

, [1] where is the dielectric constant of the vacuum ( 27 Mathematically, for any two beads (charged or neutral ones) i and j, u vdw (r ij ) is given by , [2] where σ ij (= R i + R j ) is the separation distance of two amino acids i and j at contact. For instance, σ ij for the pair VAL-GLU is 7.2Å (= R VAL + R GLU , where R VAL = 3.4Å and R GLU = 3.8Åsee ref. 53 ). The possibility to use different sizes for these beads allows the incorporation of non-J o u r n a l P r e -p r o o f specific contributions from the hydrophobic effect in the model. 43 This should preserve the macromolecular hydrophobic moments 61 and contributes to guide a correct docking orientation at short separation distances. 27 The term ε LJ regulates the strength of the attractive forces in the system. 27, 42, 43 Typically, ε LJ is assumed to be universal for any biomolecular system and equals to 0.124 kJ/mol. 42 62 Conversely, the ??-lactoglobulin-lactoferrin complexation seems to be overestimated by the usual value of ε LJ . 43 Consequently, our research strategy has been to adopt the consensus value of 0.05k B T (= 0.124 kJ/mol) for ε LJ . This also implies that the outcomes should be interpreted with relative caution bearing in mind all the intrinsic approaches assumed in the modeling. The direct impact is seen in the free energy derivatives as discussed later at the results section.

Combining Eqs. (1) and (2), the total system's interaction energy for a given configuration can be written as:

, [3] where are amino acid positions and is their total number. This includes both charged and neutral beads. that is different than other general protein-protein interfaces, 65 etc.]. 27, 55 Such facts contribute to shift the canonical view of the -lock and key‖ (with a clear focus on the protein surface) to a broader definition that led to the -electrostatic epitopes‖ (EE) concept. 27 This means that inner titratable groups (not only the ones at the epitope-paratope interface) can also participate in the interplay of interactions with Abs.

The EEs are the core idea of the PROCEEDpKa method 27 where pKa shifts are used to identify the key amino acids responsible for a host-pathogen association. It applies the fact that the location of these shifts is a practical mean to probe molecular interactions as before demonstrated. 67 Moreover, this can be easily measured during computer simulations of a proteinprotein complexation. The predictive properties of this powerful tool have been previously i) statistically analyzed for flaviviruses, ii) compared to other bioinformatic tools (that often ignore that pH and ionic strength can drastically affect the complexation process) and iii) discussed in details in a preceding work. 27 The capacity of this method to test EE for specific mAbs makes it even more appealing for the present study where four known mAbs should be investigated. For the sake of convenience, predicted EE for the studied systems were graphically compared at the sequence level. The pairwise sequence alignments were generated by the server EMBOSS Needle 68 with default settings. were not reported in Tian's work. 8 In contrast to these results, another recent study 20 advocated that SARS-CoV-2 has greater binding affinity for ACE2 than SARS-CoV-1. Even so, both the present theoretical and previously reported experimental data do agree that SARS-CoV-1 S RBD and SARS-CoV-2 S RBD have similar attraction to the ACE2. This high binding affinity implies that all human organs rich on ACE2 (oral and nasal mucosa, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain) 76 Note that the simulations were performed with a single RBD protein in the absence of the full structure of the S protein and the others two chains of the homotrimeric S glycoprotein. This brings with it the evidence that the other structural parts of the S1 subunit, the S2 subunit and the two other chains are not essential for the individual pair of RBD-ACE2 complexation (assuming that the targeted-RBD has no steric hindrance as discussed above). Also, this observation supports the argument that the dissociation of the S1 subunit complexed with ACE2 can happen without the interruption of the infection. This also allows the S2 subunit to transit from a metastable prefusion to its post-fusion state as a second step in the viral infection. 58, 78 At pH 4.6 which is closer to the low pH that occur outside the cell, 23 However, we prefer to interpret such small differences as tendencies of the system. Based on previous studies 27, 42, 43, 54, 60 where we have observed either that the computed complexation was weaker than the experimental measurements or too much stronger, we feel safer to use this data (and the others discussed below) in relative terms (i.e. comparing different situations). This allows us to successfully predict experimental observations respecting the limits of such CG models. 27, 42, 43, 54 The similarities between their free energy minima are relatively amplified at pH 4.6 (??βw min =0.07 as seen above) in comparison with pH 7.0 where βw min is −1.11 and −1.06 (??βw min =0.05). Both viral S RBD proteins have their affinities to ACE2 slightly raised when pH is increased from the acid to the neutral regime. This somewhat higher binding affinity at neutral pH suggests that the role of pH for RBD proteins constantly under debate at the literature 22, 25, 73, 80 might not be so critical for the infection. It also reinforces the possibility that the viral cell invasion is not a pH-dependent process. Indeed, it seems that pH is more relevant for the next steps to continue the viral infection and not at the first entry level. This possible non pH-depend process might increase the opportunities for the SARS viruses to easily infect cells and therefore to contribute to its high infectivity. This might limit the use of chloroquine as an efficient drug against COVID-19 since its first action is to increase the cell pH. At least a neutral pH solution will not prevent the binding of the RBD to ACE2. On the opposite as it can favor this affinity. Moreover, the work of Tian and co-authors suggested that only for CR3022 it was experimentally measured a reasonable binding. 8 The combination of these information could indicate that a threshold of −0.67K B T for βw min can be used to better refine the theoretical binding predictions of these macromolecular complexations (i.e. all viral protein-protein systems with a J o u r n a l P r e -p r o o f value of βw min smaller than −0.67K B T are expected to experience binding in vivo at least). Table   S1 summarizes the values of βw min given between parenthesis. It should be noted that the attraction between the S RBD proteins and ACE2 (βw min equals to −1.02 and −0.95 for SARS-CoV-1 and SARS-CoV-2, respectively) is always stronger than what was calculated to any studied mAb including to the CR3022 (βw min equals to −0.85 and −0.79 for SARS-CoV-1 and SARS-CoV-2, respectively) for both SARS viruses (see table S1 ). The same tendency was experimentally verified. 8 It was measured by BLI assay a K D of 6.3 nM for the J o u r n a l P r e -p r o o f binding of CR3022 to SARS-CoV-2 S RBD which corresponds to a fraction of 0.41 of the K D measured for the binding of ACE2 to the same RBD (K D equals to 15.2nM). 8

Next, we explored basic physical chemical aspects that could offer a simple and quick reasoning to understand the above free energy results and eventually be used as descriptors to scan agrees with the previous free energy analyses (see above). As large is A, larger is the attractive van der Waals interactions that can overcome the charge-charge repulsion. This can also explain why m396 (that is smaller and has roughly half of A) is less attracted to the RBD proteins even being slightly less positively charged (Z equals to 2.7 at pH 4.6) than the others (Z~5-6).

Similarly, this is the physical reason to understand the stronger binding affinity that ACE2 (A=25,290Å 2 ) has to the S RBD proteins. Although ACE2 (Z=5.4) and F26G19 (Z=5.8) have similar Zs, the molecular surface of ACE2 is ca. 2.5 times larger than F26G29 (A=10,120Å 2 ).

We tested the van der Waals (vdw) contribution comparing ??w(r) for CR3022 and m396 in a model where all electrostatic interactions where completely switched off and only vdw interactions are considered. This test-case system is shown in Figure S2 . It can be seen that m396

in this hypothetical test does have a weaker binding affinity to SARS-CoV-2 S RBD protein in comparison to CR3022 confirming the arguments above presented.

Combining the findings above reported with a theoretical alanine scanning scheme employed to determine the contribution of specific titratable group to the complexation process, we identified The binding affinity of this computer-designed molecule was tested. The calculated ??w(r) for this new fragment of mAb is given in Figure 9 . As it can be seen, CR3022' is now able to bind with an equivalent binding affinity to what was observed for the SARS-CoV-1S RBD-CR3022

system. βw min decreased from −0.79 to −0.85 recovering the value found for the SARS-CoV-1 S RBD-CR3022 case − see Figure 7 and Table S1 . Therefore, this is a promising designed mAb candidate to be carefully and systematically examined in further experimental assays. It can also be used as a template to design functional peptides and/or other inhibitors for preventing the interactions between the virus and ACE2.

J o u r n a l P r e -p r o o f 

To refine this analysis at the sequence level, the PROCEEDpKa method 27 -AWERKKISN‖ for SARS-CoV-1 and -AWNRKRISN‖ for SARS-CoV-2) indicating that the same biological interface was explored by the two viral RBD proteins in spite of their structural differences. The RBD protein responsible for COVID-19 clearly has more titratable residues interacting with ACE2 than its precursor. This observation suggests that its binding to ACE2 might be less specific than what happens for SARS-CoV-1. As such, the presence of an Ab may not completely block the SARS-CoV-2 S RBD-ACE2 interaction. In general, as seen in this for SARS-CoV-2). CR3022 perturbed more titratable groups: 27 for SARS-CoV-1 and 33 for SARS-CoV-2 (22% more). Using a different criterion to determine the epitopes, Yuan and coauthors also observed an increase of 15% in the number of titratable residues at the epitope for SARS-CoV-2. 88 Taking into account what was hypothesized for ACE2 above, this might be an addition contribution to improve the capability of this mAb to interact and inhibit the RBD proteins. In fact, the experimental work of Tian and colleagues 8 do show that the CR3022 binding to SARS-CoV-2 S RBD is not affected by ACE2. This might be the molecular basis for this behavior. We are careful with the use of stronger statements here due to the limitation of the theoretical approach. Several additional issues remain to be further investigated.

Finally, we compared the EE predictions for CR3022' (34 aa) with CR3022 (33 aa) interacting with SARS-CoV-2 S RBD protein − see Figure 11 . The predicted EEs for the interaction with CR3022' are essentially the same ones observed for CR3022 (27 common aa Figure 11 . Electrostatic epitopes. Primary sequence of the SARS-CoV-2 S RBD with the estimated antigenic regions (shown in blue) for CR3022 and CR3022' by the electrostatic method. Data for CR3022 is the same shown in Figure 10 . All other details are also as in Figure   10 .

Free energies of interactions were calculated for several molecular complexes involving the RBD of SARS-CoV-1 and 2 spike proteins. The present theoretical results confirmed that both RBD proteins have similar binding affinity to the ACE2 as previously reported in experimental studies. This is observed at both acid and neutral pH regimes which probably indicates that the medium pH is not so relevant for the beginning of the viral cell invasion. pH seems to be more important for the next steps of the viral infection and not at the first entry level. This has a direct implication for the drug development since the proposal of some like chloroquine is to raise cell pH.

Analyzing the interactions between these RBD proteins and the SARS-CoV-1 S RBD specific neutralizing mAbs (80R, F26G19, m396, CR3022) allowed us to reproduce the experimental results. The only mAb with measured affinities for the SARS-CoV-2 S RBD protein by BLI J o u r n a l P r e -p r o o f assay was CR3020 8 which was also the one with higher affinity quantified in the present theoretical study. Moreover, we could map their electrostatic epitopes and identify that all mAbs tend to share the same titratable residues, and they are like the residues involved in the interaction with ACE2. However, the RBD protein responsible for COVID-19 clearly has more titratable residues interacting with ACE2 than its precursor suggesting that its binding to ACE2 might be less specific. This can explain the general difficulty that mAbs can experience to completely block the SARS-CoV-2 S RBD-ACE2 interaction.

Charge-charge interactions were found to be a good simple descriptor for a fast screening to the designing of improved mAb for diagnostics, therapeutics and vaccines. Our theoretical approach, while still being further developed, has identified three amino acids substitution that can increase the binding affinity of CR3022 to the RBD protein responsible for the present pandemic. These results can contribute to guide the design of new functional and high specific mAbs providing a cost-and-time-effective computational framework towards the development of better diagnostic strategies and an effective treatment and/or vaccine for COVID-19. 

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Constant-PH Hybrid Nonequilibrium Molecular Dynamics−Monte Carlo Simulation Method

Stabilized Coronavirus Spikes Are Resistant to Conformational Changes Induced by Receptor Recognition or

Fast Coarse-Grained Model for RNA Titration

Protein-RNA Complexation Driven by the Charge Regulation Mechanism

Hydrophobic Moments and Protein Structure

Integrating All-Atom and Coarse-Grained Simulations-Toward Understanding of IDPs at Surfaces

Anisotropic Interactions in Protein Mixtures: Self Assembly and Phase Behavior in Aqueous Solution

Polyspecificity, and Heterospecificity of Antibody-Antigen Recognition

Antigen-Antibody Interface Properties: Composition, Residue Interactions, and Features of 53 Non-Redundant Structures

Don't Blame It All on Antibodies -The Need for Exhaustive Characterisation, Appropriate Handling, and Addressing the Issues That Affect Specificity

Computationally Mapping PKa Shifts Due to the Presence of a Polyelectrolyte Chain around Whey Proteins

A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins

Receptor-Binding Domain as a Target for Developing SARS Vaccines

Renin-Angiotensin System in Human Coronavirus Pathogenesis

Activation of Viruses by Host Proteases

Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence

Receptor and Viral Determinants of SARS-Coronavirus Adaptation to Human ACE2

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

Tissue Distribution of ACE2 Protein, the Functional Receptor for SARS Coronavirus. A First Step in Understanding SARS Pathogenesis

High Expression of ACE2 Receptor of 2019-NCoV on the Epithelial Cells of Oral Mucosa

Cryo-EM Structure of the SARS Coronavirus Spike Glycoprotein in Complex with Its Host Cell Receptor ACE2

Host Cell Proteases: Critical Determinants of Coronavirus Tropism and Pathogenesis

PH-Induced Alterations in the Fusogenic Spike Protein of Semliki Forest Virus

Exploring Designability of Electrostatic Complementarity at an Antigen-Antibody Interface Directed by Mutagenesis

PH-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus Is Mediated by the Spike Glycoprotein and Enhanced by Dendritic Cell Transfer through DC-SIGN

PH-Dependent Conformational Flexibility of the SARS-CoV Main Proteinase (Mpro) Dimer: Molecular Dynamics Simulations and Multiple X-Ray Structure Analyses

Effect of PH and Temperature on the Infectivity of Human Coronavirus 229E

Anti-Severe Acute Respiratory Syndrome Coronavirus Spike Antibodies Trigger Infection of Human Immune Cells via a PH-and Cysteine Protease-Independent Fc R Pathway

Electrostatics in Macromolecular Solutions. In Food Colloids

Cryo-EM Structure of the 2019-NCoV Spike in the Prefusion Conformation

A Highly Conserved Cryptic Epitope in the Receptor-Binding Domains of SARS-CoV-2 and SARS-CoV