Molecular Simulation

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Epitopes mapped onto SARS-CoV-2 receptor-

binding motif by five distinct human neutralising
antibodies

Lucas J. Gutiérrez, Rodrigo D. Tosso, M. Natalia C. Zarycz, Ricardo D. Enriz &

Héctor A. Baldoni

To cite this article: Lucas J. Gutiérrez, Rodrigo D. Tosso, M. Natalia C. Zarycz, Ricardo
D. Enriz & Héctor A. Baldoni (2022): Epitopes mapped onto SARS-CoV-2 receptor-
binding motif by five distinct human neutralising antibodies, Molecular Simulation, DOI:
10.1080/08927022.2022.2111421

To link to this article: https://doi.org/10.1080/08927022.2022.2111421

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MOLECULAR SIMULATION
https://doi.org/10.1080/08927022.2022.2111421

Epitopes mapped onto SARS-CoV-2 receptor-binding motif by five distinct human

neutralising antibodies
a,b a,b a a,b b,
Lucas J. Gutiérrez , Rodrigo D. Tosso , M. Natalia C. Zarycz , Ricardo D. Enriz and Héctor A. Baldoni
c

a
Multidisciplinary Institute of Biological Research (IMIBIO-SL. CONICET), San Luis, Argentina; bFaculty of Chemistry, Biochemistry and Pharmacy,
National University of San Luis, San Luis, Argentina; cInstitute of Applied Mathematics of San Luis (IMASL. CONICET), San Luis, Argentina

ABSTRACT ARTICLE HISTORY

An Immune complex formation is mediated through intermolecular interactions between proteins and Received 4 April 2022
antigens. In the present study, in silico methods were used to locate, identify and provide valuable Accepted 30 July 2022
structural and energetic information that describes the dominant and energetically stabilising
KEYWORDS
interactions found at the epitope-paratope interface of selected human antibodies, isolated from Spike protein; pandemic;
convalescent COVID-19 patients. These antibodies are directed toward the SARS-CoV-2 receptor- COVID 19; epitope-paratope
binding domain (RBD, i.e. residues 333–526). The analyzed immune complexes have shown strong in interactions; monoclonal
vitro neutralising activity against SARS-CoV-2 infection by targeting the receptor-binding motif (RBM, antibodies
i.e. residues 437–508) within the viral SARS-CoV-2 spike RBD. Our data shows that residues Tyr449,
Leu455, Phe456, Ala475, Phe486, Gln493, Gln498, Asn501, Gly502 and Tyr505 exhibit strong epitope
capability. On another hand, the analyzed paratopes show a large repertoire of residues to recognise
RBM epitopes by both the VH and VL chains within complementary determining regions (CDR), which
would make it difficult to bind RBM to the human ACE2 cell receptor by residue interactions
competition. Undoubtedly, our findings provide valuable structural and energetic information to those
previously obtained through experimental crystallographic studies, and provides new insights about
the binding interface that could provide a structural basis for rational epitope-based vaccinology.

1. Introduction represent the fundamental structural unit helpful for protec-

Coronaviruses are a family of viruses that can cause mild ill-
nesses such as the common cold [1], serious pneumonia such
as severe acute respiratory syndrome (SARS) [2] or the Middle
East respiratory syndrome (MERS) [3]. In late 2019, a new cor-
onavirus was identified as the cause of a disease outbreak first
reported in Wuhan, China [4–6]. This virus is now known as
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-
2), being the etiological agent for coronavirus disease 2019
(COVID-19) [7]; whose pathophysiology is being understood
[8]. In March 2020, the World Health Organization (WHO)
declared that the COVID-19 outbreak is a pandemic. This
virus has spread rapidly throughout the world and has infected
more than 586.6 million people, and so far more than 6.4
million people are confirmed to have died globally [9,10].
Although vaccines typically require years of research and testing
before reaching the clinic, there are currently several vaccines
approved for use in various countries [11–15]. Parallelly, new
potential drugs and targets are continuously being evaluated
to prevent, mitigate or improve the treatment of this pandemic;
however, there is still much to be studied [16–18].
Many viral structural proteins have an intrinsic capacity to
produce antibodies against the viruses from which they are
derived because they show a great capacity for the presentation
of antigenic determinants (i.e. epitopes). Commonly, epitopes
tion, screening, and choice of therapeutic and purposeful
monoclonal antibodies [19–21]. Interestingly enough, antigens
that are flexible, highly dense, or carry out multivalent epitopes
induce a wide range of antibodies. Consequently, most patho-
gens have developed strategies to evade immunity by present-
ing epitopes to the immune system that can readily undergo
antigenic variation, while hiding highly conserved sites that
are essential for protein stability-activity function [22–24].
Recently it has been possible to identify, isolate and crystal-
ise potent neutralising antibodies (nAbs) from convalescent
COVID-19 patients, which provide powerful protection
against SARS-CoV-2 infection when tested in animals and cul-
tured human cells. The finding of these antibodies set the stage
for possible clinical treatments and vaccination to prevent
COVID-19. Moreover, such antibodies could be administered
to patients in the early stage of COVID-19 to reduce the effect
of the virus and protect against the disease, or can also be used
to provide temporary protection for healthcare workers, the
elderly, immunosuppressed and others who respond poorly
to traditional vaccines [25]. These specific antibodies showed
that they could block the virus attachment to the host cells
by targeting the S1 subunit of the SARS-CoV-2 spike protein.
It is worth noting that the S1 subunit contains the RBD
responsible for mediating contacts to the human host cell
receptor angiotensin-converting enzyme 2 (hACE2) through

CONTACT Héctor A. Baldoni hbaldoni@unsl.edu.ar

Supplemental data for this article can be accessed online at https://doi.org/10.1080/08927022.2022.2111421.
© 2022 Informa UK Limited, trading as Taylor & Francis Group
2 L. J. GUTIÉRREZ ET AL.

its RBM [26]. As previously shown, the S1 subunit of the spike two steps of relaxation were carried out. The relaxation was
protein is a major immunisation target that contains multiple started by applying 20,000 steps of steepest-descent minimis-
RBD and non-RBD epitopes and has guided the design of viral ation in the presence of a 20 kcal/molÅ2 restraint on the solute
vaccines and antigens [27–31]. Nevertheless, the development backbone carbons alfa. This minimisation procedure was then
of effective antiviral vaccines requires detailed and descriptive repeated for another 20,000 steps with a 2 kcal/molÅ2 restraint
studies of such epitopes because the host immune response to on the solute backbone carbons alfa. An isochoric-isothermal
this new coronavirus is not fully understood [13,32–34]. While (NVT) ensemble was set to equilibrate the temperature of
biochemical and biophysical experiments can identify such the system at 298 K by Langevin dynamics using a 2 ps−1 col-
epitopes, they are time-consuming, expensive, and fraught lision frequency during 1 ns simulation, and left at 298 K for
with limitations. Therefore, computational that can locate 4 ns. The heating phase was followed by an adjustment of
and describe stabilising and disrupting intermolecular inter- the density in the isobaric-isothermal (NPT) ensemble during
actions that hold protein–protein complexes together can a 5 ns. The structure obtained after the NPT dynamics was
complement experimental finding on many different ways used as the starting conformation for the production phase.
[35]. In this sense, in silico approaches provide a powerful Long-range electrostatic interactions were treated by the par-
complement for experimentalists [36,37]. ticle-mesh Ewald method. Short-range non-bonded inter-
Taking advantage of previous experimental crystallographic actions were truncated with a 9 Å cutoff. All bonds involving
findings here we employ molecular simulations and end-point hydrogen atoms were constrained by the SHAKE method
binding free energy calculations [38,39] to identify, analyze allowing a time step of 2 fs for the integration of the equations
and provide valuable structural and energetic information to of motion. Snapshots of the systems were collected in time
describe the epitope-paratope interactions displayed between intervals of 5 ps along the trajectories for further analysis.
selected human antibodies obtained from convalescent In this work, we identified interface residues contributing
COVID-19 patients, which target the RBD of the SARS- the most to binding free energy by applying the end-point
CoV-2 spike protein. The selected antibodies showed strong free energy decomposition approach at the Poisson-Boltz-
neutralising activity against SARS-CoV-2 infection by binding mann (PB) and the Generalized Born (GB) approximations
the RBD at its RBM, responsible for contacting hACE2 recep- [38,39]. To increase the precision of these calculations and
tor, since the antibodies that do not bind to RBD or are promote conformational sampling the production stage was
directed to non-RBD epitopes on spike protein all show carried out running four independent replicas for each
poor neutralisation potencies [40]. immune complex with simulation length limited to 120 ns,
and the average binding free energies were calculated. Genhe-
den et al. [51] have confirmed this procedure to be effective.
2. Methods The end-point mm-pb(gb)sa method was used in a hierarchi-
The experimental crystal coordinates of selected immune com- cal strategy, details of this method have been presented else-
plexes deposited in the Protein Data Bank, entries 7BZ5 [41], where [51–53] and its accuracy have been recently reviewed
7C01 [42], 7BYR [43], 7BWJ [44], and 6XE1 [45] were [39,54,55]. This protocol was used within the one-trajectory
retrieved as structural templates for our study. All dynamics approximation, extracting equidistant snapshots of each
and free energy calculations were performed with Amber20 replica trajectories and averaging over snapshots. In brief,
package [46] and the force field ff19sb [47] was used to binding free energy (ΔGbinding) resulting from the immune
describe the immune complexes. A modified simulation pro- complex formation would be expressed as:
cedure published elsewhere was applied [48–50]. In brief,
RBD + nAb ⇔ RBD:nAb (1)
each immune complex was soaked in a box of TIP3P water
molecules with a margin of 10 Å. Before the MD simulations, where RBD:nAb is the immune complex formed by the RBD of
the SARS-CoV-2 viral spike protein and its cognate neutralis-
ing antibody (nAb) association, and can be expressed as the
Table 1. Binding energy components (kcal/mol) calculated by mm/gbsa and
mm/pbsa methods. sum of two components, gas-phase binding free energy
Immune complex (DEgas ) and solvation free energy (DGsol ):
Method Contribution 7BZ5 7C01 6XE1 7BYR 7BWJ
DGbinding = DEgas + DGsol − TDS (2)
mm/gbsa
ΔEvdw −104.8 −116.1 −96.91 −93.9 −61.2
ΔEele −169.5 −155.8 −107.59 −78.5 68.8
DEgas = DEelec. + DEvdW + DEint. (3)
ΔGGB 211.0 211.9 155.80 122.7 −38.5
ΔGnp −16.3 −17.0 −14.46 −12.3 −8.0 where ΔEele, ΔEvdW and ΔEint represent the electrostatic, vdW
ΔGele,tot 41.5 56.1 48.21 44.2 30.3 energies, and internal energies that is bond, angle and dihedral
ΔGnp,tot −121.0 −133.1 −111.37 −106.2 −69.2 energies, respectively.
ΔGbind −79.5 −77.0 −63.76 −62.0 −38.9
mm/pbsa DEgas and DGsol accounts for the ensemble-averaged RBD:
ΔEvdw −104.8 −116.1 −96.91 −93.9 −61.2 nAb enthalpic interaction energy change, and term (−TDS)
ΔEele −169.5 −155.8 −107.59 −78.5 68.8 represent the change in conformational entropy associated
ΔGPB 209.5 205.2 158.36 118.7 −38.9
ΔGnp −18.1 −17.9 −16.29 −14.6 −9.8 with the complex formation. Solvation free energy (DGsol ) is
ΔGele,tot 40.0 49.5 50.77 40.7 29.9 obtained by:
ΔGnp,tot −122.8 −134.0 −113.20 −108.4 −71.1
ΔGbind −82.8 −84.6 −62.44 −68.2 −41.2 DGsol = DGGB(PB) + DGnp (4)

Figure 1. (Colour online) Interaction energy decomposition analysis. The x-axis
denotes the RBD residue sequence and the y-axis denotes the interaction energy
contribution (ΔG, kcal/mol) to the immune complex formation. (a) 7BZ5, (b) 7C01,
(c) 6XE1.
where DGGB(PB) is the electrostatic solvation free energy
obtained by solving either GB or PB equation. The nonpolar
contribution (DGnp ) was evaluated by ΔGnp = α×SASA–β,
where SASA was the solvent accessible surface area. Sphere
probes with a radius of 1.4 Å determined molecular surface.
Per-residue binding energy decomposition analysis was
performed as described elsewhere [56,57]. The energy contri-
bution of each residue to the binding energy is given by
DGresidue = DEelec. + DEvdW + DGPB(GB) + DGnp (5)
ΔGPB(GB) is the free energy due to the solvation process of the
polar contribution calculated using GB (Generalized Born)
[38,39] or PB (Poisson–Boltzmann) [58] equations. Most of
the force field and solvation model combinations overestimate
MOLECULAR SIMULATION 3
Figure 2. (Colour online) Interaction energy decomposition analysis. The x-axis
denotes the RBD residue sequence, and the y-axis denotes the interaction energy
contribution (ΔG, kcal/mol) to the immune complex formation. (a) 7BYR, (b)
7BWJ.
the salt bridge interactions. However, combination of the
amber force field and igb8 solvent model was extensively tested
for protein interaction simulations and showed a significant
improvement in overall performance [59,60]. Thus, we
selected the igb8 solvent model for the binding energy and
per-residue binding energy decomposition analysis. Only
those RBD and nAb residues showing strong contribution to
the binding (–ΔG > 1 kcal/mol) were included in the epitope
and paratope discussion. Residues that contributed with a |
ΔG| higher than ∼1.5 kcal/mol in the per residue energy
decomposition of the paratope were subjected to compu-
tational alanine scanning mutagenesis (ASM) by single alanine
mutation [61]. Experimentally, hotspots may be located via
traditional or shotgun alanine mutations, through identifying
large binding free energy change from the mutation [62]. In
the same way, ASM was performed to determine the interface
hotspots and binding free energy difference, before (i.e. wild
type protein) and after the mutation (i.e. mutant protein), as
ΔΔGASM = ΔGmutant – ΔGwt. We choses the single-trajectory
approach to generate the alanine mutant trajectories as the
single-trajectory approach benefits from error cancellation
and can offset the insufficient sampling of conformational
space [63]. In addition, it was shown that for ASM studies
the single-trajectory approach is more efficient and accurate
than the multiple-trajectory approach [64–66]. Furthermore,
the ratio of the dissociation constants, r = Kdmutant /Kdwt
between the wild type (Kdwt ) and mutant (Kdmutant ) complex
was determined from r = e DDGb /RT [49], this ratio provided
4 L. J. GUTIÉRREZ ET AL.

an estimated of the effect of each alanine substitution on each after the resulting complex formation [66–68]. In fact, this
selection as a change in free energy relative to that of the wild behaviour has been previously proposed and is a well-accepted
type protein. scenario for antibody–antigen interactions [69–71]. In short,
although long-range interactions such as electrostatic inter-
actions (ΔGele,tot) are essential for the highly cooperative
3. Results and discussions
stabilisation of the epitope-paratope binding, favourable non-
The immune complexes PDB code: 7BZ5 [41], 7C01 [42], polar interactions (ΔGnp,tot) drive the formation of the
7BYR [43], 7BWJ [44], and 6XE1 [45] were selected from cor- immune complexes analyzed (see Table 1). These results are
onavirus antibody database [25] considering their ability to in complete agreement with previous physicochemical analysis
neutralise SARS-CoV-2 infection by in vitro assays [41–45]. on interaction forces operating between complementary epi-
All mentioned immune complexes shoved strong neutralising topes and paratopes, which showed that medium – and
activity against SARS-CoV-2 infection by targeting the RBM, long-range specific attractive forces were mainly of hydro-
within the RBD of the viral SARS-CoV-2 spike protein, phobic nature [71].
responsible for the main contacts of hACE2 receptor [26].
Conversely, it must be noted that antibodies that do not target
3.1. RBM epitopes
RBD or are directed to non-RBD epitopes, show poor neutral-
isation potencies [27,40]. Global stability and flexibility of the The RBM binding epitopes were inferred from the free energy
immune complexes were evaluated measuring the root mean decomposition spectra of the analyzed immune complexes.
square deviation (rmsd) and root mean square fluctuation Free energy decomposition spectrum for immune complexes
(rmsf) taken the crystallographic coordinates as references, 7BZ5, 7C01 and 6XE1 are depicted in Figure 1, while Figure
Figures S1 and S2, respectively. All immune complexes reach 2 shows the spectrum obtained for immune complexes 7BYR
rmsd lower than 2.5 Å along the simulation time. Additionally, and 7BWJ. Based on free energy decomposition analysis
as observed in the rmsf profiles (Figure S2) the simulated con- (Figures 1 and 2) it might be observed that key residues
formational ensembles clearly show that the systems are more unevenly spread within RBM drives the formation of the
dynamic than their crystallographic forms. immune complexes. In other words, the cognate antibody
From our results, rmsd shows that the simulated dynamics bind a discontinuous and far apart epitopes in the RBM pri-
does not deviate significantly from its starting coordinates mary sequence but assembled by 3D folding (i.e. confor-
while rmsf shows adequate flexibility giving a picture of mational epitopes). These epitopes consist of interface
which parts of the protein are moving and how much each residues within the targeted RBD and are mostly located out-
part of the protein has changed over the course of the simu- side α or β secondary structural elements.
lated assembly. Considering the aforementioned results,
rmsd and rmsf in our study serves as indirect evidence sup- Table 2. RBM epitopes targeted by the indicated antibodies.
porting the global quality and folding stability of our system, Immune complex
and all dynamics features follow a consistent trend. RBM epitopes a 7BZ5 c 7C01 c 6XE1 c
7BYR c 7BWJ c
In order to address the identification of contact residues Val445 VH
involved in the epitope-paratope interaction and evaluate its Gly446 VH
binding energy contributions, the simulated dynamics were Gly447 VH
Asn448 VH
followed by the end-point free energy decomposition Tyr449b VH VH
approach at the PB and GB approximations [38,39]. To Asn450 VH
increase precision of our calculations, four independent repli- Leu452 VH VH
Tyr453 VH
cas of production were performed for each system, and average Leu455b VH VH VH
binding free energies were calculated. Total binding energy Phe456b VH VH VH
and energy components are summarised in Table 1. Arg457 VH VH
Tyr473 VH VH VH
The immune complexes showed favourable net binding Ala475b VH VH VH
affinity ΔGbinding (i.e. ΔGele,tot+ΔGnp,tot), within the range of Gly476 VH VH
−38.9 to −79.5 kcal/mol, and within −41.2 to −84.6 kcal/mol Ser477 VH VH VH
Thr478 VH
at GB and PB approximation, respectively. As listed in Table Val483 VL
1, van der Waals (ΔEvdw), electrostatic (ΔEele) and nonpolar Glu484 VL
solvation energies (ΔGnp) terms provide the most favourable Gly485 VH
Phe486b VH VH VH VH
contributions to the binding. Contrarily, the polar solvation Asn487 VH VH VH
energies (ΔGPB/GB) impair the binding energy needed for the Tyr489 VH VH VH
immune complexes formation. Indeed, Table 1 shows that Phe490 VH VL
Gln493b VH
the favourable ΔGbinding arise from favourable nonpolar inter- Ser494 VH
actions ΔGnp,tot (i.e. ΔEvdW+ΔGnp) for all analyzed immune Gln498b VH
complexes despite unfavourable electrostatic contribution Asn501b VL VL
Gly502b VL
ΔGele,tot (i.e. ΔEele+ΔGPB/GB). Usually, the electrostatic contri- Tyr505b VL VL VL VH
bution disfavours the binding of ligands to their receptor This work. b Key interacting residues (in bold) between SARS-CoV-2 RBM and
a
because the unfavourable change in solvation electrostatics is hACE2 receptor identified by Andújar et al. [48]. c Interaction mainly mediated
not fully compensated for by the favourable electrostatics by antibody VH or VL chain.

Figure 3. (Colour online) Schematic representations for snapshots of the analyzed immune complexes showing the binding pose and the RBM hotspots. RBD (in blue
ribbon with RBM highlighted in yellow). Antibody VH chain (red Cα stick) and VL chain (green Cα stick). (a) 7BZ5, (b) 7C01, (c) 6EX1, and (d) 7BWJ.
Antibodies represented by Immune complexes 7BZ5,
7C01 and 6XE1 target RBM conformational epitopes invol-
ving residues Leu455, Phe456, Tyr473, Ala475, Ser477,
Phe486, Asn487 and Tyr505 with different energy strength
(Figure 1). Besides the mentioned residues, Thr478 and
Gly502 also play a significant role in the immune complex
7BZ5 (Figure 1), while residue Tyr489 is involved in com-
plexes 7C01 and 6XE1 (Figure 1). Notably, residues
Arg457, Gly476, Asn501 are targeted in complexes 7BZ5
and 7C01 (Figure 1). In turn, residue Tyr453 is only
involved in complex 6XE1 (Figure 1).
On the other hand, antibodies represented by immune
complexes 7BYR and 7BWJ target quite different RBM discon-
tinuous epitopes from those recognised by complexes 7BZ5,
7C01 and 6XE1. Our results show that residues Tyr449,
Leu452, and Phe490 are epitopes for both 7BYR and 7BWJ
complexes (Figure 2). However, while residues Gly446,
Gly485, Phe486, Tyr489, Glu493 Ser494, Gln498 and Tyr505
are involved in the 7BYR complex formation (Figure 2), resi-
dues Val445, Gly447, Asn448, Asn450, Val483 and Glu484
are targeted in complex 7BWJ (Figure 2). It should be noted
that RBM residues Phe486, Tyr489 and Tyr505 targeted by
complex 7BYR (Figure 2) are also targeted by the previously
analyzed complexes 7BZ5, 7C01 and 6XE1 as seen in Figure 1.
Remarkably, residue Glu484 plays a slightly unfavourable
role in the binding in complex 7BYR (Figure 2), but contrarily
MOLECULAR SIMULATION 5
its contribution is favourable in complex 7BWJ (Figure 2). It
should be noted that the five analyzed immune complexes
(Figures 1 and 2) target residues Phe486, Tyr489 and Tyr505
on RBM. The above-mentioned RBM hotspots are depicted
in Figure 3 and succinctly summarised in Table 2.
It is important to highlight that RBM residues Tyr449,
Leu455, Phe456, Ala475, Phe486, Gln493, Gln498, Asn501,
Gly502 and Tyr505 were described playing a key role in
RBM:hACE2 interface interactions [48,72,73]. Thus, it could
be argued that RBM-epitopes masking by their cognate anti-
bodies would mediate neutralisation by direct competition
against the interacting partners in concordance with in vitro
assays [31,35–37]. However, other requirements regarding
antigen–antibody stoichiometry, kinetics, and epitope-para-
tope steric hindrances are relevant factors in many virus neu-
tralisation [74–77].
It is worth noting that, only some hotspots residues listed in
Table 2, among them Leu452, Tyr489, Asn501, and Tyr505,
were pointed out, and in fact verified, to have high chances
to mutate towards significantly more transmissible or infec-
tious SARS-CoV-2 strains [78,79]. In fact, SARS-CoV-2 var-
iants of concern that have been gaining traction around the
world include mutations of RBM residues Leu452, Glu484
and Asn501 as listed in Table 2. Moreover, analysis of the
data collected by Mercatelli et al. [80] shows that from 1205
genetically identified SARS-CoV-2 polymorphs involving
6 L. J. GUTIÉRREZ ET AL.
Figure 4. (Colour online) Interfacial hydrogen bonds (HBs) events (y-axis) versus timeline (x-axis) within all four replicas analyzed from continuous trajectories. HBs are
plotted as blue bars when formed (value 1), and black bar when broken (value 0). (a) immune complex 7BZ5, (b) immune complex 7C01, (c) immune complex 6XE1.
single-nucleotide mutations (SNM) into the spike protein, 141
of such SNM involve the RBM, from the latter 62 SNMs are
associated with hotspots identified in this work (Table S1). It
has been proposed that these RBM mutants would be able to
evade anti-spike vaccines-induced immunity, or COVID vac-
cines-induced immune response could favour RBM mutant’s
evolution by mutant escape [73,81–88]. Actually, these studies
make us aware of the possibility of reinfection with antigeni-
cally distinct variants and may herald a reduced efficacy of cur-
rent spike-based vaccines [89–93].
To better understand the molecular interactions that
occurs at the binding interface in these immune complexes,
the interface hotspots and non-hotspots residues that partici-
pate in persistent energetically favourable and strong inter-
actions, i.e. hydrogen bonds and salt bridges, were
analyzed and depicted in Figures 4 and 5, and listed in
Table 3.
Indeed, the hydrogen bonds analysis of the immune com-
plexes pinpoints a larger number of more persistent hydrogen
bonds and salt bridges at the interface of 7C01 > 7BZ5 ≈ 7BWJ
> 6XE1 ≈ 7BWJ. The residues establishing the most important
hydrogen bond interactions have been confirmed also by
experimental studies [41–45]. Hydrophobic interactions also
play an important role in stabilising the RBM:hACE2 interface.
Our analysis confirms that hydrophobic residues Phe, Leu,
Val, and Ile at the RBM are especially important role in inter-
act with paratopes as it has the highest frequency of partici-
pation in the interfacial hydrophobic interactions.
Hydrophobic residues (Ala, Met, Pro, Trp and Tyr) are
under-represented amino acids among the RBM interfacial
hydrophobic contact residues. Interfacial RBM:mAbs hydro-
phobic interactions results are presented in Table 4.
In summary, our findings indicate that in the analyzed
immune complexes contact binding epitopes are formed by
energetically critical hotspots located into the RBM within
the RBD. Moreover, even though structural epitopes might
be located neighbouring the RBM, as has been proposed else-
where [94–96], no critical residues for interface recognition or
 MOLECULAR SIMULATION 7

Figure 5. (Colour online) Interfacial hydrogen bonds (HBs) events (y-axis) versus timeline (x-axis) within all four replicas analyzed from continuous trajectories. HBs are
plotted as blue bars when formed (value 1), and black bar when broken (value 0). (a) Immune complex 7BYR, (b) immune complex 7BWJ.

binding were found outside the RBM region. Furthermore, it 3.2. Paratopes localisation, identification and
seems clear from our results that masking the identified mutations
RBM hotspots by their cognate neutralising antibodies would
Antibody engineering requires ascertaining precisely the resi-
hinder the binding to the hACE2 cell receptor through direct
dues that contact and interact with the antigen hotspots to
competition of interface residues, preventing the binding of
adjust precisely binding affinity and specificity of the anti-
SARS-CoV-2 RBD to hACE2.
body–antigen interaction [97–100]. Therefore, we located
and identified residues in each contacting regions of the anti-
bodies that contribute energetically to the RBM binding, for a
Table 3. Interfacial hydrogen bonds and salt bridges (in bold). deeper understanding of the recognition of RBM hotspots by
Hydrogen Bonds a the selected antibodies.
RBM 7BZ5 % occupancy
Asn487 Arg97 87.4
Asn501 Ser30 21.5 3.2.1. Immune complex 7BZ5
Ser477 Asp1 18.4 Our results show that three contacting regions on the variable
Asp420 Ser56 13.3 heavy chain (VH) domain, namely H1: Phe27, Ile28, Ser31,
Arg408 Asp1 10.6 b
7C01 Asn32 and Tyr33; H2: Tyr52, Ser53, Gly54 and Ser56; H3:
Asn487 Arg97 85.8 Arg97, Tyr100 and Asp103 (the later opposes the binding);
Asn460 Ser56 11.8 and two contacting regions on the variable light chain (VL)
Tyr421 Ser56 34.8
Tyr473 Ser31 22.3 domain, namely L1: Gly28, Ile29, Ser30 and Tyr32; L3:
Tyr505 Tyr32 13.2 Leu91, Asn92 and Tyr94 represent hotspots involved in med-
6XE1 iating favourable energy interactions with the RBM (Figure S3
Asn487 Arg97 78.9
Tyr453 Tyr252 21.3 and Table S2). Our ASM analysis reveals that the affinity of this
Tyr88 Tyr33 11.5 antibody is strongly affected by alanine mutation. Alanine
7BYR mutation of the mentioned hotspots disfavours the binding
Gln493 Thr53 48.6
Gln493 Asn54 31.3 free energy from ∼1 to ∼6 kcal/mol, which gives high dis-
Gln493 Asn54 13.2 sociation constant of up to ∼4 fold, Table S2.
7BWJ
Glu484 Arg112 89.6 b
Glu484 Tyr24 16.4 3.2.2. Immune complex 7C01
Note: Only events with occupancy time >10% of the simulation time are shown. A detailed analysis shows that H1: Phe27, Thr28, Ser31, Asn32
a
Hydrogen-Acceptor distance cutoff dH … A ≤ 3.0 Å and the Donor-H-Acceptor
angle cutoff αDHA ≥ 135.0 degrees.
and Tyr33; H2: Tyr52, Ser53, Gly54, Ser56 and Phe58; H3:
b
Donor-Acceptor distance cutoff dDA ≤ 3.0 Å and the Donor-Acceptor angle cutoff Arg97, Leu99, Pro100, Met101 and Tyr102; L3: Tyr92, Thr94
αDA ≥ 135.0 degrees. and Pro95, mediates the hotspots RBM contacting sites. In
8 L. J. GUTIÉRREZ ET AL.
Table 4. Interfacial hydrophobic interactions a.
RBM 7BZ5 RBM 7C01
Phe486 Val2 (73) Phe456 Pro100 (89) Phe456
Phe456 Ala99 (40) Leu455 Pro100 (66) Leu455
Phe486 Phe27 (12) Phe456 Met101 (44) Phe456
Phe486 Val104 (10) Phe486 Val1 (35) Phe486
Leu455 Met101 (33)
Phe486 Phe27 (32)
Phe456 Leu99 (15)
a
Hydrophobic interactions are defined as sidechains interactions between any non-polar amino acids (Ala, Ile, Leu, Met, Phe, Pro, Val, Tyr, Trp) when their distance is
less than 5Å. Only events with occupancy time ≥10% over the simulation time are shown. The % occupancy is show in parenthesis. RBM (residues 437-508). Antibody
VH chain (residues 1-216).
other words, three contacting regions on the VH chain, and
one contacting region on the VL chain, mediate the inter-
actions with the RBM (Figure S4). ASM analyses suggest that
antibody affinity is strongly affected by alanine mutation
(Table S2). Alanine substitutions of the specified hotspots dis-
favour the binding free energy from ∼1 to ∼7 kcal/mol, which
implies high dissociation constant of up to ∼5 fold.
3.2.3. Immune complex 6XE1
Our data indicate that three contacting regions on the variable
heavy chain (VH) domain, namely H1: Val27, Ile28 and Tyr33;
H2: Tyr52 and Ser56; H3: Arg97, Leu99, Val101 and Ser102;
and two contacting regions on the variable light chain (VL)
domain, namely L1: Ser28, Ser30 and Tyr33; and L3: Gly93
mediates the contacting with the RBM (Figure S5). Antibody
affinity is disrupted in a significant way when these hotspots
are alanine mutated. As shown in Table S2, alanine mutation
of the implied hotspots disfavours binding free energy from
∼1 to ∼6 kcal/mol, implying high dissociation constant of
up to ∼4 fold.
3.2.4. Immune complex 7BYR
Three contacting regions on the VH chain, namely H1: Tyr53,
Asn54, Thr55, Gly56 and Pro58; H2: Val69 and Leu72; H3:
Gly102, Ser103 and Ser104 mediates the interactions with
RBM. Notably, the VL chain does not contact the RBM
(Figure S6). In addition, one non-CDR region (residues
Phe29 and Tyr32) has a role in RBM binding interactions
(Figure S6). Alanine mutations of these hotspots disrupt anti-
body affinity in a significant way. Our data show that alanine
mutation disfavours the binding free energy from ∼1 to ∼4
kcal/mol, which suggests high dissociation constant of up
to ∼3 fold, see Table S2.
3.2.5. Immune complex 7BWJ
The analyses demonstrate that two contacting regions on the
VH chain, i.e. H1: Tyr27 and Ser31; and H3: Ile103, Val105,
Val106, Pro107 and Arg112 make direct contact with RBM.
Whereas VL chain hardly mediate contacts with RBM through
L1: Tyr22, Asn23 and Tyr24. We found that, Glu52 located at
L2 opposes binding (Figure S7). ASM results revealed that,
except for Glu52 substitution, all proposed alanine mutation
strongly affects the RBM binding free energy. Alanine
mutation of the mentioned hotspots disfavours the binding
free energy from ∼2 to ∼6 kcal/mol, indicating high dis-
sociation constant of up to ∼4 fold as listed in Table S2.
RMB 6XE1 RBM 7BYR RBM 7BWJ
Leu99 (34) Phe490 Trp105 (90) Phe490 Val106 (91)
Val101 (26) Phe486 Phe29 (47) Leu452 Ile103 (52)
Val101 (16) Phe456 Phe29 (20) Phe490 Pro107 (51)
Val2 (10) Val445 Val69 (18) Phe490 Ile103 (25)
Leu492 Trp105 (13) Phe490 Val105 (18)
Leu452 Val105 (17)
Leu492 Val105 (13)
Ile472 Pro107 (10)
Our results reveal that the contacting residues in the ana-
lyzed antibodies, those that make relevant energetic contri-
butions to the binding interaction (i.e. contact paratopes)
(Table S2) account for about one-quarter of the side-chains
buried at the structural paratopes interface. Such residues
strongly contribute to the interaction energy and are located
close to the centre of the structural paratope. These results
are in complete agreement with those of Kunik et al.
[101,102], derived by comparison between the consensus
regions of structural paratopes from structural alignments of
a non-redundant set of known antibody–antigen complexes.
In addition, we found that electrostatic interactions partly
modulate the immune complexes formation because
mutations at Tyr, Ser and Arg disfavour the binding free
energy by increasing the dissociation constants ratio in about
2.0–4.5 fold (Table S2). These results are in concordance
with previous findings supporting the intrinsic contributions
of Tyr, Ser, Gly and Arg residues to the affinity and specificity
in the recognition of many antigens (Birtalan et al., 2008).
Noteworthy, Tyr, Ser and Gly residues are common in anti-
body mutation hotspots and often mutated during somatic
hypermutation [103–105].
In short, it is important to note that our results demon-
strate that some contacting residues located at CDR at the
immune complexes interface play a dominant role in the
effective interface interaction with the SARS-CoV-2 RBM.
Along these lines, the identified contacting residues at the
paratopes interface shows a great variety of interatomic inter-
actions to hook and mask RBM epitopes, making it difficult
for RBM to bind to the hACE2 receptor through competitive
interactions between residues [48,72,106]. On the other hand,
in silico alanine mutations show that substitution of these
important contact residues are not tolerated and worsen the
antibodies affinity.
4. Conclusions
By performing all-atom MD simulations, we have investigated
the atomic-level fine definition of the epitope recognised by
human neutralising antibodies toward SARS-CoV-2 RBD,
which is an important aspect to be considered for possible
development in epitope-based vaccinology. The presented
results undoubtedly give additional support and provide valu-
able structural and energetic information to those obtained else-
where. Our findings reveal that residues Tyr449, Leu455,
Phe456, Ala475, Phe486, Gln493, Gln498, Asn501, Gly502 and

Tyr505 located at the RBM within the RBD region possess
strong epitope capabilities, at least for the immune complexes
here analyzed. Computational analysis of the antibodies’ para-
topes shows a vast collection of interaction in both of its variable
regions of light (VL) and heavy chains (VH) on each CDR.
Unfortunately, the analyzed antibodies do not allow us to pro-
pose a biospecific consensus paratope, reason why the rate of
epitope-paratope specificity and selectivity might be involved
in vivo or in vitro response. As we demonstrate here, masking
RBM hotspots by their cognate neutralising antibodies would
make it difficult to bind to the hACE2 cell receptor through
direct competition of interactions between interface residues,
thereby abolishing the binding of SARS-CoV-2 RBD to its target
hACE2 cell receptor in human cells. The different residues
identified in the analyzed paratopes show that COVID-19
recovered patients have a great repertory to develop
biospecific antibodies, a feature that significantly involves the
specificity success, reason why further epitopes-paratopes
characterisation eventually may be important in COVID-19
immunotherapy.
Acknowledgments
In memory of Prof. Dr. Esteban Adrián Jáuregui (1929-2022) and Prof.
PhD. Imre Gyula Csizmadia (1932-2022), were great inspirations for
the authors as well as for several generations of students and scientists
in our University.
Funding
Grants from Universidad Nacional de San Luis (UNSL-Argentina) and
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-
Argentina) PIP 090CO, partially supported this work.
CRediT authorship contribution statement
Contributions are defined using CRediT for standardised con-
tribution descriptions. L.J.G., R.D.T., and M.N.C.Z.: method-
ology, data curation, visualisation. R.D.E.: resources, writing-
review & editing. H.A.B. (leader): methodology, conceptualis-
ation, formal analysis, funding acquisition, investigation, pro-
ject administration, supervision, writing-original draft,
writing-review & editing.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
Grants from Universidad Nacional de San Luis (UNSL-Argentina) and
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-
Argentina) PIP 090CO, partially supported this work.
ORCID
Lucas J. Gutiérrez http://orcid.org/0000-0003-2776-517X
Rodrigo D. Tosso http://orcid.org/0000-0003-0798-2003
M. Natalia C. Zarycz http://orcid.org/0000-0001-9838-5778
Ricardo D. Enriz http://orcid.org/0000-0002-4846-9452
Héctor A. Baldoni http://orcid.org/0000-0003-2688-1697
MOLECULAR SIMULATION 9
References
[1] Makela MJ, Puhakka T, Ruuskanen O, et al. Viruses and bacteria in
the etiology of the common cold. J Clin Microbiol. 1998;36
(2):539–542.
[2] Drosten C, Gunther S, Preiser W, et al. Identification of a novel
coronavirus in patients with severe acute respiratory syndrome.
N Engl J Med. 2003;348(20):1967–1976.
[3] Zaki AM, van Boheemen S, Bestebroer TM, et al. Isolation of a
novel coronavirus from a man with pneumonia in Saudi Arabia.
N Engl J Med. 2012;367(19):1814–1820.
[4] Chan JF, Yuan S, Kok KH, et al. A familial cluster of pneumonia
associated with the 2019 novel coronavirus indicating person-to-
person transmission: a study of a family cluster. Lancet.
2020;395(10223):514–523.
[5] Huang C, Wang Y, Li X. Clinical features of patients infected with
2019 novel coronavirus in Wuhan, China. Lancet. 2020;395
(10223):497–506.
[6] Zhu N, Zhang D, Wang W, et al. A novel coronavirus from
patients with pneumonia in China, 2019. N Engl J Med.
2020;382(8):727–733.
[7] Khan S, Siddique R, Bai Q, et al. Coronaviruses disease 2019
(COVID-19): causative agent, mental health concerns, and poten-
tial management options. J Infect Public Health. 2020b;13
(12):1840–1844.
[8] Bohn MK, Hall A, Sepiashvili L, et al. Pathophysiology of COVID-
19: mechanisms underlying disease severity and progression.
Physiology (Bethesda). 2020;35(5):288–301.
[9] John Hopkins Coronavirus Resource Center. Johns Hopkins
University & Medicine; [cited 2022 Aug 2]. Availabel from:
https://coronavirus.jhu.edu/us-map.
[10] Sanche S, Lin YT, Xu C, et al. High contagiousness and rapid
spread of severe acute respiratory syndrome coronavirus 2.
Emerg Infect Dis. 2020;26(7):1470–1477.
[11] Diamond MS, Pierson TC. The challenges of vaccine development
against a new virus during a pandemic. Cell Host Microbe. 2020;27
(5):699–703.
[12] Eichler HG, Cavaleri M, Enzmann H, et al. Clinical trials for
COVID-19: can we better Use the short window of opportunity?
Clin Pharmacol Ther. 2020;108(4):730–733.
[13] Lee WS, Wheatley AK, Kent SJ, et al. Antibody-dependent
enhancement and SARS-CoV-2 vaccines and therapies. Nat
Microbiol. 2020;5(10):1185–1191.
[14] Pottegard A, Kurz X, Moore N, et al. Considerations for pharma-
coepidemiological analyses in the SARS-CoV-2 pandemic.
Pharmacoepidemiol Drug Saf. 2020;29(8):825–831.
[15] Thanh Le T, Andreadakis Z, Kumar A, et al. The COVID-19 vac-
cine development landscape. Nat Rev Drug Discov. 2020;19
(5):305–306.
[16] Plavec Z, Pöhner I, Poso A, et al. Virus structure and structure-
based antivirals. Curr Opin Virol. 2021;51:16–24.
[17] Raj K, Kaur K, Gupta GD, et al. Current understanding on molecu-
lar drug targets and emerging treatment strategy for novel corona-
virus-19. Naunyn Schmiedebergs Arch Pharmacol. 2021;394
(7):1383–1402.
[18] Patten JJ, Keiser PT, Gysi D, et al. Identification of druggable host tar-
gets needed for SARS-CoV-2 infection by combined pharmacological
evaluation and cellular network directed prioritization both in vitro
and in vivo. Available from: https://ssrn.com/abstract = 4038525.
[19] Heffron AS, McIlwain SJ, Amjadi MF, et al. The landscape of anti-
body binding in SARS-CoV-2 infection. PLoS Biol. 2021;19(6):
e3001265.
[20] Luan B, Huynh T, Cheng X, et al. Targeting proteases for treating
COVID-19. J Proteome Res. 2020;19(11):4316–4326.
[21] Shu T, Ning W, Wu D, et al. Plasma proteomics identify bio-
markers and pathogenesis of COVID-19. Immunity. 2020;53
(5):1108–1122. e5.
[22] Finlay BB, McFadden G. Anti-Immunology: evasion of the host
immune system by bacterial and viral pathogens. Cell. 2006;124
(4):767–782.
10 L. J. GUTIÉRREZ ET AL.
[23] Ernst JD. Antigenic variation and immune escape in the MTBC.
Adv Exp Med Biol. 2017;1019:171–190.
[24] Vossen MT, Westerhout EM, Soderberg-Naucler C, et al. Viral
immune evasion: a masterpiece of evolution. Immunogenetics.
2002;54(8):527–542.
[25] Raybould MIJ, Kovaltsuk A, Marks C, et al. CoV-AbDab: the cor-
onavirus antibody database. Bioinformatics. 2021;37(5):734–735.
[26] Shang J, Ye G, Shi K, et al. Structural basis of receptor recognition
by SARS-CoV-2. Nature. 2020;581(7807):221–224.
[27] Amrun SN, Lee CY, Lee B, et al. Linear B-cell epitopes in the spike
and nucleocapsid proteins as markers of SARS-CoV-2 exposure
and disease severity. EBioMedicine. 2020;58:102911.
[28] Kanduc D. From anti-SARS-CoV-2 immune responses to
COVID-19 via molecular mimicry. Antibodies (Basel). 2020;9
(3):33.
[29] Kong WH, Zhao R, Zhou JB, et al. Serologic response to SARS-
CoV-2 in COVID-19 patients with different severity. Virol Sin.
2020;35(6):752–757.
[30] Speiser DE, Bachmann MF. COVID-19: mechanisms of vacci-
nation and immunity. Vaccines (Basel). 2020;8(3):404.
[31] Zhang LX, Miao SY, Qin ZH, et al. Preliminary analysis of B- and
T-cell responses to SARS-CoV-2. Mol Diagn Ther. 2020;24
(5):601–609.
[32] Arvin AM, Fink K, Schmid MA, et al. A perspective on potential
antibody-dependent enhancement of SARS-CoV-2. Nature.
2020;584(7821):353–363.
[33] Desheva YA, Mamontov AS, Nazarov PG. Contribution of anti-
body-dependent enhancement to the pathogenesis of coronavirus
infections. AIMS Allergy and Immunology. 2020;4(3):50–59.
[34] Wen J, Cheng Y, Ling R, et al. Antibody-dependent enhancement
of coronavirus. Int J Infect Dis. 2020;100:483–489.
[35] Roterman-Konieczna I. Identification of ligand binding site and
protein-protein interaction area. Netherlands: Springer; 2013.
[36] Chain B, Greiff V, Textor J, et al. Editorial: methods and appli-
cations of computational immunology. Front Immunol.
2019;10:2818.
[37] Clementi N, Mancini N, Castelli M, et al. Characterization of epi-
topes recognized by monoclonal antibodies: experimental
approaches supported by freely accessible bioinformatic tools.
Drug Discov Today. 2013;18(9-10):464–471.
[38] Wang E, Sun H, Wang J, et al. End-Point binding free energy cal-
culation with MM/PBSA and MM/GBSA: strategies and appli-
cations in drug design. Chem Rev. 2019a;119(16):9478–9508.
[39] Weng G, Wang E, Chen F, et al. Assessing the performance of
MM/PBSA and MM/GBSA methods. 9. prediction reliability of
binding affinities and binding poses for protein-peptide com-
plexes. Phys Chem Chem Phys. 2019;21(9):10135–10145.
[40] Rogers TF, Zhao F, Huang D, et al. Isolation of potent SARS-CoV-
2 neutralizing antibodies and protection from disease in a small
animal model. Science 2020;369(6506):956-963.
[41] Wu Y, Wang F, Shen C, et al. A noncompeting pair of human neu-
tralizing antibodies block COVID-19 virus binding to its receptor
ACE2. Science. 2020;368(6496):1274–1278.
[42] Shi R, Shan C, Duan X, et al. A human neutralizing antibody tar-
gets the receptor-binding site of SARS-CoV-2. Nature. 2020;584
(7819):120–124.
[43] Cao Y, Su B, Guo X, et al. Potent neutralizing antibodies against
SARS-CoV-2 identified by high-throughput single-cell sequencing
of convalescent patients’ B cells. Cell. 2020;182(1):73–84.
[44] Ju B, Zhang Q, Ge J, et al. Human neutralizing antibodies elicited
by SARS-CoV-2 infection. Nature. 2020;584(7819):115–119.
[45] Hurlburt NK, Seydoux E, Wan YH, et al. Structural basis for
potent neutralization of SARS-CoV-2 and role of antibody
affinity maturation. Nat Commun. 2020;11(1):5413.
[46] Case DA, Aktulga HM, Belfon K, et al. Amber. San Francisco:
University of California; 2021.
[47] Tian C, Kasavajhala K, Belfon KAA, et al. ff19SB: amino-acid-
specific protein backbone parameters trained against quantum
mechanics energy surfaces in solution. J Chem Theory Comput.
2020;16(1):528–552.
[48] Andújar SA, Gutiérrez LJ, Enriz RD, et al. Structure, interface stab-
ility and hot-spots identification for RBD(SARS-CoV-2):
hACE2 complex formation. Molecular Simulation 2021;47
(17):1443–1454.
[49] Gutierrez LJ, Andújar SA, Enriz RD, et al. Structural and func-
tional insights into the anti-BACE1 Fab fragment that recognizes
the BACE1 exosite. J Biomol Struct Dyn. 2014;32(9):1421–1433.
[50] Siano A, Garibotto FF, Andújar SA, et al. Molecular design and
synthesis of novel peptides from amphibians skin acting as inhibi-
tors of cholinesterase enzymes. J Pept Sci. 2017;23(3):236–244.
[51] Genheden S, Ryde U. The MM/PBSA and MM/GBSA methods to
estimate ligand-binding affinities. Expert Opin Drug Discov.
2015;10(5):449–461.
[52] Sun H, Li Y, Tian S, et al. Assessing the performance of MM/PBSA
and MM/GBSA methods. 4. accuracies of MM/PBSA and MM/
GBSA methodologies evaluated by various simulation protocols
using PDBbind data set. Phys Chem Chem Phys. 2014;16
(31):16719–16729.
[53] Rastelli G, Del Rio A, Degliesposti G, et al. Fast and accurate pre-
dictions of binding free energies using MM-PBSA and MM-GBSA.
J Comput Chem. 2010;31(4):797–810.
[54] Izadi S, Harris RC, Fenley MO, et al. Accuracy comparison of gen-
eralized born models in the calculation of electrostatic binding free
energies. J Chem Theory Comput. 2018;14(3):1656–1670.
[55] Wang E, Weng G, Sun H, et al. Assessing the performance of the
MM/PBSA and MM/GBSA methods. 10. Impacts of enhanced
sampling and variable dielectric model on protein-protein inter-
actions. Phys Chem Chem Phys. 2019b;21(35):18958–18969.
[56] Khan A, Tahir Khan M, Saleem S, et al. Structural insights into the
mechanism of RNA recognition by the N-terminal RNA-binding
domain of the SARS-CoV-2 nucleocapsid phosphoprotein.
Comput Struct Biotechnol J. 2020a;18:2174–2184.
[57] Xu D, Si Y, Meroueh SO. A computational investigation of small-
molecule engagement of hot spots at protein-protein interaction
interfaces. J Chem Inf Model. 2017;57(9):2250–2272.
[58] Rocchia W. Poisson-boltzmann equation boundary conditions for
biological applications. Math. and Comput. Model. 2005;41
(10):1109–1118.
[59] Robinson MK, Monroe JI, Shell MS. Are AMBER force fields and
implicit solvation models additive? A folding study with a balanced
peptide test Set. J Chem Theory Comput. 2016;12(11):5631–5642.
[60] Zeng J, Li Y, Zhang JZ, et al. Examination of the quality of various
force fields and solvation models for the equilibrium simulations
of GA88 and GB88. J Mol Model. 2016;22(8):177.
[61] Bradshaw RT, Patel BH, Tate EW, et al. Comparing experimental
and computational alanine scanning techniques for probing a pro-
totypical protein-protein interaction. Protein Eng Des Sel. 2011;24
(1-2):197–207.
[62] Weiss GA, Watanabe CK, Zhong A, et al. Rapid mapping of
protein functional epitopes by combinatorial alanine scanning.
Proc Natl Acad Sci USA. 2000;97(16):8950–8954.
[63] Aldeghi M, Bodkin MJ, Knapp S, et al. Statistical analysis on the
performance of molecular mechanics Poisson-Boltzmann surface
area versus absolute binding free energy calculations: bromodo-
mains as a case study. J Chem Inf Model. 2017;57(9):2203–2221.
[64] Martins SA, Perez MA, Moreira IS, et al. Computational alanine
scanning mutagenesis: MM-PBSA vs TI. J Chem Theory
Comput. 2013;9(3):1311–1319.
[65] Massova I, Kollman PA. Computational alanine scanning To
probe protein−protein interactions: a novel approach to
evaluate binding free energies. J. Am. Chem. Soc. 1999;121
(36):8133–8143.
[66] Moreira IS, Fernandes PA, Ramos MJ. Computational alanine
scanning mutagenesis an improved methodological approach. J
Comput Chem. 2007;28(3):644–654.
[67] Novotny J, Bruccoleri RE, Davis M, et al. Empirical free energy cal-
culations: a blind test and further improvements to the method. J
Mol Biol. 1997;268(2):401–411.
[68] Sharp KA. Electrostatic interactions in hirudin-thrombin binding.
Biophys Chem. 1996;61(1):37–49.

[69] Nguyen MN, Pradhan MR, Verma C, et al. The interfacial charac-
ter of antibody paratopes: analysis of antibody-antigen structures.
Bioinformatics. 2017;33(19):2971–2976.
[70] Qiu T, Xiao H, Zhang Q, et al. Proteochemometric modeling of the
antigen-antibody interaction: new fingerprints for antigen, anti-
body and epitope-paratope interaction. PLoS One. 2015;10(4):
e0122416.
[71] Van Oss CJ. Hydrophobic, hydrophilic and other interactions in
epitope-paratope binding. Mol Immunol. 1995;32(3):199–211.
[72] Ghorbani M, Brooks BR, Klauda JB. Critical sequence hotspots for
binding of novel coronavirus to angiotensin converter enzyme as
evaluated by molecular simulations. J Phys Chem B. 2020;124
(45):10034–10047.
[73] Tsai KC, Lee YC, Tseng TS. Comprehensive deep mutational scan-
ning reveals the immune-escaping hotspots of SARS-CoV-2 recep-
tor-binding domain targeting neutralizing antibodies. Front
Microbiol. 2021;12:698365.
[74] Della-Porta AJ, Westaway EG. A multi-hit model for the neutral-
ization of animal viruses. J Gen Virol. 1978;38(1):1–19.
[75] Klasse PJ, Sattentau QJ. Occupancy and mechanism in antibody-
mediated neutralization of animal viruses. J Gen Virol. 2002;83
(Pt 9):2091–2108.
[76] Platt EJ, Gomes MM, Kabat D. Kinetic mechanism for HIV-1 neu-
tralization by antibody 2G12 entails reversible glycan binding that
slows cell entry. Proc Natl Acad Sci USA. 2012;109(20):7829–7834.
[77] Reading SA, Dimmock NJ. Neutralization of animal virus infectiv-
ity by antibody. Arch Virol. 2007;152(6):1047–1059.
[78] Chen J, Wang R, Wang M, et al. Mutations strengthened SARS-
CoV-2 infectivity. J Mol Biol. 2020a;432(19):5212–5226.
[79] Greaney AJ, Starr TN, Gilchuk P, et al. Complete mapping of
mutations to the SARS-CoV-2 spike receptor-binding domain
that escape antibody recognition. Cell Host & Microbe. 2021;29
(1):44–57.
[80] Mercatelli D, Giorgi FM. Geographic and genomic distribution of
SARS-CoV-2 mutations. Front Microbiol. 2020;11:1800.
[81] Chen LL, Lu L, Choi CY, et al. Impact of Severe Acute Respiratory
Syndrome Coronavirus 2 (SARS-CoV-2) Variant-Associated
Receptor Binding Domain (RBD) Mutations on the
Susceptibility to Serum Antibodies Elicited by Coronavirus
Disease 2019 (COVID-19) Infection or Vaccination. Clin Infect
Dis. 2022a;74(9):1623–1630.
[82] Chen X, Chen Z, Azman AS, et al. Neutralizing antibodies against
SARS-CoV-2 variants induced by natural infection or vaccination:
a systematic review and pooled meta-analysis. Clin Infect Dis.
2022;74(4):734–742.
[83] Ikegame S, Siddiquey MNA, Hung CT, et al. Neutralizing activity
of Sputnik V vaccine sera against SARS-CoV-2 variants. Nat
Commun. 2021;12(1):4598.
[84] Starr TN, Czudnochowski N, Liu Z, et al. SARS-CoV-2 RBD anti-
bodies that maximize breadth and resistance to escape. Nature.
2021;597(7874):97–102.
[85] Vasireddy D, Vanaparthy R, Mohan G, et al. Review of COVID-19
variants and COVID-19 vaccine efficacy: what the clinician should
know? J Clin Med Res. 2021;13(6):317–325.
[86] Wibmer CK, Ayres F, Hermanus T, et al. SARS-CoV-2 501Y.V2
escapes neutralization by South African COVID-19 donor plasma.
Nat Med. 2021;27(4):622–625.
[87] Wilson P, Changrob S, Fu Y, et al. Cross neutralization of emer-
ging SARS-CoV-2 variants of concern by antibodies targeting dis-
tinct epitopes on spike. mBio. 2021;12(6):e0297521.
[88] Yoo JH. What We Do know and Do Not Yet know about COVID-
19 vaccines as of the beginning of the year 2021. J Korean Med Sci.
2021;36(6):e54.
MOLECULAR SIMULATION 11
[89] Alenquer M, Ferreira F, Lousa D, et al. Signatures in SARS-CoV-2
spike protein conferring escape to neutralizing antibodies. PLoS
Pathog. 2021;17(8):e1009772.
[90] Jeyanathan M, Afkhami S, Smaill F, et al. Immunological consider-
ations for COVID-19 vaccine strategies. Nat Rev Immunol.
2020;20(10):615–632.
[91] Koopman JS, Simon CP, Getz WM, et al. Modeling the population
effects of escape mutations in SARS-CoV-2 to guide vaccination
strategies. Epidemics. 2021;36:100484.
[92] Tregoning JS, Flight KE, Higham SL, et al. Progress of the COVID-19
vaccine effort: viruses, vaccines and variants versus efficacy, effective-
ness and escape. Nat Rev Immunol. 2021;21(10):626–636.
[93] Van Egeren D, Novokhodko A, Stoddard M, et al. Risk of rapid
evolutionary escape from biomedical interventions
targeting SARS-CoV-2 spike protein. PLoS One. 2021;16(4):
e0250780.
[94] Bian C, Zhang X, Cai X, et al. Conserved amino acids W423 and
N424 in receptor-binding domain of SARS-CoV are potential tar-
gets for therapeutic monoclonal antibody. Virology. 2009;383
(1):39–46.
[95] Chen W-H, Hotez PJ, Bottazzi ME. Potential for developing a
SARS-CoV receptor-binding domain (RBD) recombinant protein
as a heterologous human vaccine against coronavirus infectious
disease (COVID)-19. Human Vaccines & Immunotherapeutics.
2020b;16(6):1239–1242.
[96] Zhou D, Duyvesteyn HME, Chen CP, et al. Structural basis for the
neutralization of SARS-CoV-2 by an antibody from a convalescent
patient. Nat Struct Mol Biol. 2020;27(10):950–958.
[97] Jian JW, Chen HS, Chiu YK, et al. Effective binding to protein
antigens by antibodies from antibody libraries designed with
enhanced protein recognition propensities. MAbs. 2019;11
(2):373–387.
[98] Schreiber G. Protein–protein interaction interfaces and their func-
tional implications. In: Roy S., Fu H. editors. Protein–protein
interaction regulators. London : The Royal Society of Chemistry;
2020. p. 1–24.
[99] Sela-Culang I, Kunik V, Ofran Y. The structural basis of antibody-
antigen recognition. Front Immunol. 2013;4:302.
[100] Yu CM, Peng HP, Chen IC, et al. Rationalization and design of the
complementarity determining region sequences in an antibody-
antigen recognition interface. PLoS One. 2012;7(3):e33340.
[101] Kunik V, Ashkenazi S, Ofran Y. Paratome: an online tool for sys-
tematic identification of antigen-binding regions in antibodies
based on sequence or structure. Nucleic Acids Res. 2012a;40
(Web Server issue):W521–W524.
[102] Kunik V, Peters B, Ofran Y. Structural consensus among anti-
bodies defines the antigen binding site. PLoS Comput Biol.
2012b;8(2):e1002388.
[103] Clark LA, Ganesan S, Papp S, et al. Trends in antibody sequence
changes during the somatic hypermutation process. J Immunol.
2006;177(1):333–340.
[104] Muecksch F, Weisblum Y, Barnes CO, et al. Affinity maturation of
SARS-CoV-2 neutralizing antibodies confers potency, breadth,
and resilience to viral escape mutations. Immunity. 2021;54
(8):1853–1868.
[105] Birtalan S, Zhang Y, Fellouse FA, et al. The intrinsic
contributions of tyrosine, serine, glycine and arginine to the
affinity and specificity of antibodies. J Mol Biol. 2008;377
(5):1518–1528.
[106] Wan Y, Shang J, Graham R, et al. Receptor recognition by the
novel coronavirus from Wuhan: an analysis based on decade-
long structural studies of SARS coronavirus. J Virol. 2020;94(7):
e00127–20.