Journal of Biomolecular Structure and Dynamics

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Zika virus envelope – heat shock protein A5

(GRP78) binding site prediction

Abdo A. Elfiky & Ibrahim M. Ibrahim

To cite this article: Abdo A. Elfiky & Ibrahim M. Ibrahim (2020): Zika virus envelope – heat shock
protein A5 (GRP78) binding site prediction, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2020.1784794

To link to this article: https://doi.org/10.1080/07391102.2020.1784794

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1784794

Zika virus envelope – heat shock protein A5 (GRP78) binding site prediction
Abdo A. Elfiky and Ibrahim M. Ibrahim
Biophysics Department, Faculty of Sciences, Cairo University, Giza, Egypt
Communicated by Ramaswamy H. Sarma

ABSTRACT ARTICLE HISTORY

Recent studies reported the association of the Zika virus (ZIKV) with a stress response receptor on the Received 16 January 2020
host cell membrane that facilitates viral entry. This host receptor was the heat shock protein A5 Accepted 15 June 2020
(HSPA5), also termed glucose-regulating protein 78 (GRP78). In this study, structural bioinformatics and
KEYWORDS
molecular dynamics simulations were utilized to suggest the binding site of ZIKV envelope protein
GRP78; BiP; ZIKV envelope;
during the interaction with cell-surface GRP78. The Pep42 cyclic peptide was used as a profiler, as it protein-protein docking;
was reported earlier, to target GRP78 on the cancer cell membrane selectively. Sequence and struc- structural bioinformat-
tural alignments show that part of the ZIKV envelope protein (C308-C339 region), in addition to its ics; Pep42
cyclic nature, has somehow sequence and structural similarities to the cyclic Pep42. Three amino acids
in the ZIKV envelope were identical to those in the Pep42 peptide. Cyclic peptides dynamics are
studied, and its binding to GRP78 is predicted. Protein-protein docking is further performed to explore
the binding characteristics of the ZIKV envelope to GRP78. Results revealed that the binding was favor-
able between ZIKV envelope protein and GRP78. The docking pose revealed the involvement of the
substrate-binding domain ß of GRP78 and the domain III of the ZIKV envelope protein in viral recogni-
tion for the host-cell.

Abbreviations: ATF6: Activating Transcription Factor 6; Bip: Binding immunoglobulin protein; ER:
Endoplasmic Reticulum; CS-GRP78: Cell-surface Glucose regulated protein 78; HSPA5: Heat shock pro-
tein A5; IRE1: Inositol-requiring Enzyme 1; MDS: Molecular Dynamics Simulation; NS5: Non-Structural 5;
PDB: Protein Data Bank; PERK: Protein kinase RNA-like Endoplasmic Reticulum Kinase; RMS: Root Mean
Square; RoG: Radius of Gyration; SASA: Surface Accessible Surface Area; SBD: Substrate-binding
domain; ZIKV: Zika Virus

Introduction transmission route of ZIKV is through mosquito bites, but

ZIKV is a virus that appeared for the first time in Uganda in
the year 1947 in the Zika forest, hence termed the Zika virus
(Dick et al., 1952). Its outbreak that started in 2015 in Latin
America got a high momentum even though it wasn’t the
only reported outbreak for ZIKV (Cao-Lormeau et al., 2014;
Duffy et al., 2009; Hayes, 2009). Other outbreaks appeared in
Africa at different times but weren’t with the same wide-
spread as the Brazilian one (Campos et al., 2015). The main
the sexual transmission was reported as well (Foy et al.,
2011; Magalhaes et al., 2018; Musso et al., 2015). The symp-
toms are usually mild, like slight fever, rash, arthralgia, and
conjunctivitis. But after confirming the link between mothers
infected during pregnancy and the development of micro-
cephaly in their babies, the World Health Organization
(WHO) classified the situation as of international concern in
2016 (Mlakar et al., 2016). Besides, some neurological

CONTACT Abdo A. Elfiky abdo@sci.cu.edu.eg; aelfiky@ictp.it Biophysics Department, Faculty of Sciences, Cairo University, Giza, Egypt
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2 A. A. ELFIKY AND I. M. IBRAHIM
complications were reported after infections. ZIKV affects all
cells of the nervous system, including neurons. The neuro-
logical disorders accompanying ZIKV infection motivated
drug designers to search for an adequate drug against the
virus. Nowadays, over 84 countries confirmed ZIKV infection
(Elfiky & Elshemey, 2018). The high transmission rate was
attributed to its vector mosquito (Ades mosquitos), which
spreads worldwide (Boyer et al., 2018). ZIKV encodes ten pro-
teins, among them, is the envelope protein responsible for
virus entry (Petersen et al., 2016). Several attempts to use
antiviral drugs against ZIKV rendered good results (Elfiky,
2016; Elfiky, 2017; Elfiky & Ismail, 2018). On the other hand,
targeting the viral envelope protein (responsible for host cell
attachment and entry) will be critical for drug design or in
the development of a vaccine.
Glucose Regulating Protein 78 (GRP78), Binding immuno-
globulin protein (BiP), or Heat shock protein A5 (HSP5A) are the
names of the master chaperone protein of the unfolded protein
response (Chiappori et al., 2016). GRP78 regulates the cell
response under stress conditions (as the accumulation of
unfolded proteins) together with other chaperones in the lumen
of the Endoplasmic Reticulum (ER) (Lee, 2005; Li & Lee, 2006;
Quinones et al., 2008; Rao et al., 2002). Under normal circum-
stances, GRP78 is found in the lumen of ER bound and hence,
inactivating other enzymes responsible for cell response under
accumulated unfolded proteins. These enzymes are Activating
Transcription Factor 6 (ATF6), Protein kinase RNA-like
Endoplasmic Reticulum Kinase (PERK), and Inositol-requiring
Enzyme 1 (IRE1) (Ibrahim et al., 2019a). Under the stress of accu-
mulated unfolded proteins, the GRP78 releases the enzymes,
causing the activation of ATF6, PERK, and IRE1, which inhibit
protein synthesis and enhance refolding (Ibrahim et al., 2019a;
Shen et al., 2002). Overexpression of GRP78 is also reported
upon stress conditions. This gives a chance for GRP78 to escape
ER retention and to translocate to the cell surface. Once translo-
cated to the cell membrane, GRP78 is susceptible to pathogen
recognition and entry (viral and fungal pathogens) by its C-ter-
minal substrate-binding domain (SBD) (Elfiky, 2020a; Elfiky,
2020b; Elfiky, 2020c; Ibrahim et al., 2019b; Ibrahim et al., 2020).
Pep42 is a cyclic peptide that is reported to be a selective
binder against cell-surface GRP78 and used to carry anti-
cancer agents, Doxorubicin, to the cancer cells. The cyclic
Pep42 has 13 residues (CTVALPGGYVRVC), where Cys1 forms
a disulfide bond with Cys13 (Kim et al., 2006a). Its selectivity
is reported for its cyclic form but not the extended form.
This may be due to the rigidity of the cyclic structure of the
peptide that causes stabilization of the hydrophobic region
formed by C1, V3, A4, L5, V10, V12, and C13 that become
close to each other by the aid of the disulfide bond, making
the cyclic peptide the perfect docking platform for GRP78
SBDb (Elfiky, 2020b). Another reason that may cause the
selectivity for only the cyclic form is the degradation of the
linear peptide in vivo or immune system recognition
(Davydov & Varshavsky, 2000; White et al., 2017). The N-end
Cys in the linear peptide is degraded through the Ubiquitin
ligase E3 component N-recognin (UBR) E3 ligases after it is
subjected to oxidation and arginylation through the N-end
rule in mammalian cells. In contrast, the cyclic peptide does
not have free N-end residue (Tasaki et al., 2012). The degrad-
ation rate of linear Pep42 is predicted to be 1.2 hr (by
ProtParam software) in mammalian cells.
Different host-cell receptors were reported to interact with
ZIKV proteins. Kazmirchuk and his co-workers made one of the
previous predictions of the interaction between ZIKV mem-
brane (M) and non-structural proteins (NS3, NS5) with the host
receptor. (Kazmirchuk et al., 2017). In their work, they predicted
four human proteins to be interacted with ZIKV proteins by
using Protein-Protein Interaction Prediction Engine (PIPE) and
De Novo (Kazmirchuk et al., 2017). The first protein is
Ribonuclease T2 (RNASET2), which is related to microcephaly
(Henneke et al., 2009) and was predicted to interact with NS3
(Kazmirchuk et al., 2017). The second protein is Enolase 2
(ENO2) and was found to be linked to humans brain develop-
ment (Eriksson et al., 1998). This protein is predicted to interact
with NS5 (Kazmirchuk et al., 2017). The third and fourth pro-
teins are TNF receptor-associated factor 4 (TRAF4) and
Centrosomal protein of 63 kDa (CEP63). They interact with M
and NS3 protein (Kazmirchuk et al., 2017). A previous study by
Sujit et al. showed that cell-surface GRP78 could bind to ZIKV
envelope protein. To know the role of ZIKV infection, they over-
expressed GRP78 by increasing the temperature and by chem-
ical induction using geldanamycin, then they observed the
production of ZIKV, which was increased (Pujhari et al., 2017).
They found that, after inhibiting GRP78, the production of ZIKV
was reduced (Pujhari et al., 2017). Therefore, it was deduced
that the cell-surface GRP78 is involved in ZIKV entry by binding
to envelope protein. This protein (GRP78) was reported to act
as a receptor to other viruses’ envelope-protein such as
Japanese encephalitis virus and Dengue virus (Das et al., 2009;
Reyes-del Valle et al., 2005).
In this work, we provide a prediction of the interaction
site between ZIKV envelope protein with human GRP78. We
suggest a ZIKV envelope - GRP78 binding site based on
sequence and structural similarities between the Pep42 and
ZIKV envelope protein. Moreover, we clarify the interaction
pattern of Pep42 with GRP78. Molecular docking combined
with molecular dynamics simulations were employed to, fur-
ther, explore such binding and to refine the interacting
amino acids in the binding site.
Materials and methods
The thirteen x-ray crystallography solved structures for ZIKV
envelope protein were downloaded from the Protein Data
Bank (PDB) database (Berman et al., 2000). Both the struc-
tures (PDB) and the sequences (FASTA) were retrieved. The
removal of other chains, water, and ions from the PDB files
are carried out using PyMOL software (DeLano, 2002). The
downloaded PDB codes were: 5KVF, 5KVG, 5KVD, 5KVE,
5JHM, 5LBV, 6JEP, 5LBS, 6DFI, 5GZO, 5GZN, 5JHL and 5VIG
with a resolution range from 1.4 to 3 Å. GRP78 solved struc-
ture (PDB ID: 5E84) was downloaded and prepared for the
docking experiment. It was the only solved structure of wild-
type, full-length GRP78 bound to ATP in the open configur-
ation (Yang et al., 2015; Yang et al., 2017). Multiple Sequence
Alignment (MSA) of the sequences of ZIKV envelope and the

peptide Pep42 were performed using the T-coffee web ser-
ver of the European Bioinformatics Institute (EMBL-EBI)
(Notredame et al., 2000). ESpript 3 software was used to rep-
resent the MSA (Gouet et al., 1999). ProtParam web server of
the ExPASy bioinformatic resource portal (Garg et al., 2016)
was used to compare the Pep42 sequence to the part of the
ZIKV envelope protein sequence that we suggest it is the
binding site to GRP78.
The Pep42 3 D structure was predicted using the I-Tasser
web server (Zhang, 2008). The C-score and TM-score are
0.32 and 0.67 ± 0.13, respectively. Since the C-score ranges
from [-5,2], and a large value means more confidence in the
model, we can say that this model is good. For TM-Score, a
value > 0.5 means the model has correct topology. After
building the Pep42 model, Gauss view v5.0 software was
used to add the disulfide bond between the C1 and C13.
Then Gaussian 09 W software was used to make geometry
optimization using the UFF force field (Frisch et al., 2009;
Rappe et al., 1992). Structural alignment between each of
the 13 structures of the ZIKV envelope and the predicted
structure of Pep42 was performed with the help of the align
option of PyMOL software.
Molecular Dynamics Simulation (MDS) was performed on
both Pep42 CTVALPGGYVRVC, cyclic peptide, and the part of
the ZIKV envelope protein suggested to bind to cell-surface
GRP78 (C308: C339) CTAAFTFTKIPAETLHGTVTVEVQYAGTDGPC
for 100 ns. MDS is performed to get the different conforma-
tions cyclic peptides can take (its dynamics) since we don’t
have any solved structure for Pep42 peptide. Besides, MDS is
used to compare the dynamics of Pep42 to that of the ZIKV
envelope C308: C339 region. Additionally, 50 ns MDS of the
full-length ZIKV Envelope is performed. The dynamics were
simulated in NVT ensemble (at 310 0K) using a periodic
boundary condition of box sizes 28.7  28.7  28.7 A3 and
42.8  42.8  42.8 A3 for Pep42 and ZIKV envelope protein
(C308: C339 region), respectively. Langevin piston is used to
control the pressure of the system during the simulation
(1.01325 par). Peptides were solvated using the TIP3P water
model. At the same time, the CHARMM 36 force field is uti-
lized by NAMD software to minimize (steepest descent algo-
rithm) and equilibrate the system as well as in the
production run. Backbone-Root Mean Square Deviation
(RMSD), Per residue Root Mean Square Fluctuations (RMSF),
Radius of Gyration (RoG), Surface Accessible Surface Area
(SASA) and the number of H-bonds are calculated using
VMD TK console scripts. The clustering tool in Chimera soft-
ware is utilized to represent the different conformations of
Pep42 and ZIKV envelope (C308: C339) during the 100 ns
MDS using the implemented automated method in Chimera
software (Kelley et al., 1996; Pettersen et al., 2004).
HpepDOCK webserver was utilized to test the binding
mode of different conformations (different clusters) of the
cyclic peptides (taken during the MDS) against the GRP78
substrate-binding domain. HpepDOCK is a docking software
developed to test peptides against protein docking, and it
gives good results compared to HADDOCK in the peptide-
protein docking mode (Zhou et al., 2018).
JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 3
Full-length ZIKV envelope protein is tested against GRP78
SBDb. HADDOCK web server was used to perform protein-
protein docking using the active amino acids for ZIKV enve-
lope protein as predicted from both the MSA and the struc-
tural alignments (Dominguez et al., 2003). Active residues are
the amino acid residues from the two interacting proteins’
binding sites that take part in direct interaction with the
other protein partner in HADDOCK. The active residues from
the ZIKV envelope protein were predicted to be C308, T309,
and A311 (identical residues to that found in Pep42). GRP78
active residues (I426, T428, V429, V432, T434, F451, S452,
V457, and I459) were retrieved from the literature (Yang
et al., 2015). In both proteins, the passive amino acids were
selected to be the amino acids surrounding the
active residues.
Results and discussion
Sequence and structural alignment
Figure 1 shows the Multiple Sequence Alignment (MSA)
between the 13 solved structures of ZIKV envelope protein
and the sequence of the peptide Pep42. As mentioned
above, the cyclic form of this peptide selectively targets cell-
surface GRP78. The existence of three identical residues
(C308, T309, and A311 resemble C1, T2, and A4 in Pep42)
made the sequence identity more than 23% between the
ZIKV envelope sequences and Pep42. This value is still not
enough to support the functional similarity between Pep42
and ZIKV envelope protein. Interestingly, we found that the
ZIKV envelope C308: C339 region is cyclic. A disulfide bond
between C308 and C339 resembles the cyclic Pep42. The
secondary structure of one of the solved structures is repre-
sented at the top of the MSA, while the surface accessibility
of the residues is shown at the bottom. Green numbers
below the MSA indicate the position of the disulfide bond
C308-C339. As implicated from the MSA, the C308: C339
region has small beta strands (b1, b2, and b3) like the main
secondary structures (antiparallel b sheet) in the predicted
Pep42 model.
Figure 2A demonstrates the structural alignment of the
Pep42 3 D model, built by the I-TASSER web server, and the
13 ZIKV envelope protein solved structures (superimposed).
The Ca Root Mean Square Deviation (RMSD) from each pro-
tein structure to the peptide ranges from 2.643 to 2.882 Å.
The best match for the structural superposition against
Pep42 is the domain III of the ZIKV envelope protein. The
Pep42 model is validated against Molprobity, where 90.0% of
the residues are in the allowed region with only one residue
outlier (Williams et al., 2018).
On the other hand, the Pep42 structure is not maintained
after running MDS, as we can see in Figure 2B. This figure
shows the superposition of different four conformations of
Pep42 (representing different clusters for the 100 ns MDS
run) with the ZIKV envelope structure (PDB ID: 6JEP chain E).
Colored cartoons represent four different Pep42 peptide con-
formations (at different dynamic states); cyan (at 12.7 ns),
green (at 55.8 ns), yellow (at 81.5 ns), and magenta (at
95.2 ns), while ZIKV envelope is represented in the gray
4 A. A. ELFIKY AND I. M. IBRAHIM
Figure 1. Part of the multiple sequence alignment of all the solved structures for ZIKV envelope protein and Pep42 sequence. The PDB code for each sequence is
written in the sequence header. The alignment is made using the T-coffee web server and represented by ESpript 3 software. The red highlighted residues are the
identical residues found in both ZIKV envelope protein and the Pep42. Secondary structures are represented at the top of the MSA, while the surface accessibility
is shown at the bottom.
cartoon with the C308: C339 region in red. As shown in
Figure 2B and 2C, the C308: C339 region of the ZIKV enve-
lope is surface accessible. Additionally, there is a disulfide
bond (shown in black sticks) between C308 and C339, mak-
ing this part of the envelope protein (all the 13 solved struc-
tures) cyclic like the Pep42 peptide. The superposition
indicates the similarity between the two peptides at the
disulfide ends, due to three identical residues, C308, T309,
and A311. The best conformation for Pep42 in terms of devi-
ation from the ZIKV envelope is the conformation at 95.2 ns
(magenta) with only 0.66 Å RMSD.
Pep42 versus C308: E339 ZIKV envelope
As predicted from both MSA and structural alignment, and
since Pep42, which targets cell-surface GRP78, and ZIKV
envelope C308: C339 region are both cyclic; This part of the
ZIKV envelope may be the binding site to the cell-sur-
face GRP78.
ExPASy bioinformatics research portal ProtParam web ser-
ver (Gasteiger et al., 2005) is used to compare the sequences
of Pep42 and ZIKV envelope C308: C339 peptides. The grand
average of hydropathy (GRAVY) is 1.1 for Pep42. Yet, it is
only 0.219 in ZIKV envelope C308: C339 peptide, indicating
that despite this part of the ZIKV envelope is still hydropho-
bic, its average hydrophobicity is lower compared to the
Pep42 and hence the binding to GRP78 maybe somehow dif-
ferent. The non-polar residues contents of Pep42 is about
85%, while it is only 60% in ZIKV envelope C308: C339 pep-
tide. Figure 3 depicts the hydropathy versus the amino acids
in the two cyclic peptides. Notably, the first part of the two
cyclic peptides has the same hydropathy trend, which can
be harnessed to speculate the pattern of interaction of the
first part of the ZIKV envelope C308-C339 cyclic peptide
with GRP78.
Molecular dynamics simulation
MDS is performed for both Pep42 and the ZIKV envelope
C308: C339 peptides (PDB ID: 6JEP, Chain E) up to 100 ns.
The 100 ns MDS was enough to equilibrate the two peptide
systems, as indicated by RMSD values. Figure 4A shows the
Ca RMSD, RoG, and SASA over time for Pep42, and ZIKV
envelope C308: C339 cyclic peptides. Both systems are equili-
brated as implied from the flattened RMSD versus time of
the simulation. Pep42 is more stable compared to the larger
peptide of the ZIKV envelope. Both RoG and SASA indicate
stabilization of the simulated systems as there are little fluc-
tuations of the values of RoG ( ̴ 7 Å and 13 Å) and SASA ( ̴ 6 Å
and 5 Å) for Pep42 and ZIKV envelope C308: C339 cyclic pep-
tides, respectively. Figure 4B shows the per-residue RMSF for
both Pep42 (orange) and ZIKV envelope C308: C339 (gray)
cyclic peptides. As expected, the RMSF of the small peptide
(Pep42) is lower than the larger one (ZIKV envelope C308:
C339). The average RMSF for Pep42 is 1.97 Å ± 0.4 while it is
3.22 Å ± 0.9 in the case of ZIKV envelope C308: C339, where
the errors here are the standard deviation of the values.
Mann-Whitney U test shows a score of 26 out of 416 (multi-
plication of the number of data points), which means the
data are significantly different (McKnight & Najab, 2010). It is
expected that the C308: C339 region of the ZIKV envelope
will gain some stability when bound to the other parts of
the domain III of the ZIKV envelope. Additionally, the pres-
ence of a disulfide bond stabilizes the terminal residues that
are usually movable in linear peptides. The stabilization
effect is more apparent in Pep42 cyclic peptide (RMSF > 3 Å)
while in the larger peptide (ZIKV envelope C308: C339), the
RMSF fluctuate between 2 and 5.5 Å.
Additionally, MDS (100 ns) for the protein GRP78 in free
(blue) and bound states (with Pep42 (orange) and ZIKV enve-
lope C308: C339 (gray)) are performed using the same soft-
ware (see Figure 4C). The most fluctuating region is depicted
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 5

Figure 2. A) Structural alignment of the model for Pep42 (black cartoon) with the 13 solved structures of ZIKV envelope protein (colored cartoons). An enlarged
panel of the pep42 structure aligned with the 13 solved structures of the domain III of the ZIKV envelope protein. B) The superposition of Pep42 (four different
conformations from different clusters after 100 ns MDS) with the structure of ZIKV envelope protein (PDB ID 6JEP chain E). The Pep42 conformations are repre-
sented by the cyan (12.7 ns), green (55.8 ns), yellow (81.5 ns), and magenta (95.2 ns), while ZIKV envelope is represented in a gray cartoon with the C308: C339
region in red. Disulfides are shown in black sticks. The superposition indicates the similarity between the two peptides at the disulfide ends, due to three identical
residues, C308, T309, and A311. C) The C308: C339 region (red surface) of the ZIKV envelope (gray surface) is surface accessible, as shown in the front and back
view of the domain III (PDB ID: 6JEP chain E).
6 A. A. ELFIKY AND I. M. IBRAHIM
Figure 3. Hydrophobicity versus amino acids for Pep42 (blue) and ZIKV envelope C308: C339 (orange) cyclic peptides. The dashed lines mark disulfide bonds for
each peptide.
in red-colored cartoons (SBDa), while SBDb is encircled with
dashed-green on the GRP78 structure at the top of the fig-
ure. The corresponding residues are shown in the corre-
sponding squares on the RMSF chart. As implied from Figure
4C, the addition of the small Pep42 peptide (13 residues)
doesn’t affect the dynamics of GRP78. In comparison, the
larger (32 residues) and hence more fluctuating peptide from
ZIKV envelope affects the per residue RMSF of GRP78.
Pep42 & ZIKV envelope C308: C339 - GRP78
binding mode
The selectivity of cyclic Pep42 to cell-surface GRP78 was
reported, but the binding mode between Pep42 and GRP78
is yet to be determined (Ibrahim et al., 2019a; Kim et al.,
2006b; Yoneda et al., 2008). This study provides a proposed
binding mode between cyclic Pep42 and GRP78 using
HPEPDOCK webserver. Four different conformations of the
cyclic Pep42 peptide (representing the main four distinct
clusters of the MDS trajectories), taken at various times dur-
ing the MDS run after system equilibration, are docked into
the substrate-binding domain (SBD) of GRP78 (PDB ID: 5E84).
Figure 5A depicts the docking of these four conformations of
Pep42 cyclic peptides (yellow, magenta, cyan, green car-
toons) to GRP78 SBD (wheat cartoon). The C1 and C13 (black
sticks) and the amino acids around it are the central part of
Pep42 that form interactions with GRP78 SBD, except for the
Pep42 at 12.7 ns. We have to mention that this is the first
time to predict the binding mode between Pep42 cyclic pep-
tide and cell-surface GRP78. Table 1 summarizes the interac-
tions that formed upon docking the different conformations
of Pep42 cyclic peptides to the GRP78 substrate-binding
domain. Noteworthy, the interacted residues from Pep42 are
the residues that lie around the disulfide bond (C1-C13), resi-
dues 1, 2, 3, 5, 9, 11, and 12. This part of the peptide is the
most hydrophobic (see Figure 3) and agrees with previous
reports about the selectivity of GRP78 to the hydrophobic
patches of the unfolded proteins (Kim et al., 2006b; Yoneda
et al., 2008).
The same binding mode is found for ZIKV envelope C308:
C339 cyclic peptide (cyan cartoon in Figure 5B). We use a
conformation at 51.4 ns representing the most populated
cluster of the MDS for the peptide. The residues, F312, T315,
V328, E329, V330, and T335 near the disulfide bond (C308-
C339) are the interacting residues. Three H-bonds are estab-
lished between T315, V328, and E329 of the ZIKV envelope
C308: C339 cyclic peptide and Q449, T434, and T434 of the
GRP78 SBD, respectively. Additionally, nine hydrophobic con-
tacts are reported between F312, T315, E329, V330, and T335
from ZIKV envelope peptide and I426, T428, V429, L436,
Q449, F451 (3), and V490 from GRP78. The HpepDOCK score
(rigid docking) for the binding between ZIKV envelope C308:
C339 cyclic peptide and GRP78 is 74.3. In Figure 5B and C,
the two cyclic peptides (the most populated cluster for each
peptide) are superimposed on the SBD of GRP78 to show
how these two cyclic peptides are interacting upon docking

(see the enlarged panel of Figure 5B and C and the 90
rotated view of Figure 5B). Noticeable, the two peptides
lying superimposed to each other, while the binding mode is
mostly through the disulfide (black sticks) ends of the cyclic
peptides. Both peptide associate with T428, V429, T434,
Q449, and F451 (pink sticks) of the GRP78 SBD, but ZIKV
envelope peptide has more chance to form other interac-
tions with I426 and V490 (blue colored residues) of GRP78
SBDb through its E329 and T335 residues. The HpepDOCK
score for the top-ranked cluster of conformations of ZIKV
envelope C308: C339 cyclic peptide is 74.3.
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 7

Figure 4. A) RMSD (blue), SASA (gray) and RoG (orange) for the Pep42 (upper panel) and ZIKV envelope C308: C339 (lower panel) cyclic peptides versus time in
ns. B) RMSF for Pep42 (orange columns) and ZIKV envelope C308: C339 (gray columns). The box diagram shows the difference between the Pep42 (orange) and
ZIKV envelope C308: C339 (gray) RMSF values. C) the RMSF of GRP78 in the free state (blue) bound to Pep42 (orange), and bound to ZIKV envelope C308: C339
(gray). The highly fluctuating regions are circled in the structure and the RMSF chart.
8 A. A. ELFIKY AND I. M. IBRAHIM

Figure 5. A) Superposition of the four most populated cluster conformations of Pep42 cyclic peptide (yellow, magenta cyan, and green cartoons) after docking in
GRP78 SBDb (wheat cartoon). The interacting residues of GRP78 are labeled. B) superposition of the docked structures of Pep42 at 55.8 (magenta cartoon) and
ZIKV envelope C308: C339 region at 51.4 ns (cyan cartoon) into GRP78 (wheat cartoon) SBDb. The interacting residues are labeled and colored pink (both Pep42
and ZIKV envelope C308: C339 region) and blue (ZIKV envelope C308: C339 region only), as shown in the enlarged panel and the rotated view. C) The same as B
but with GRP78 in surface representation.
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 9

Table 1. The interactions formed between Pep42 cyclic peptides and GRP78 SBDb upon docking. The four peptide conformations are taken at different times
after clustering the coordinates to represent the most populated four conformations (after the equilibration period).
H-bonding Hydrophobic interaction
Pep42 conformation at HpepDock score number Residues from Pep42 Residues from GRP78 number Residues from Pep42 Residues from GRP78
12.7 ns 68.5 3 C1 S452 4 T2 V490
T2 S452 L5 V429
R11 S452 Y9 V429
Y9 V429
55.8 ns 94.2 4 V3 V429 3 T2 V429
Y9 Q449 V3 T428
R11 T428 R11 F451
R11 T434
81.5 ns 69.8 1 C13 S452 3 R11 T428
V12 I426
V12 F451
95.2 ns 92.9 3 C1 T456 2 Y9 V490
Y9 G454 R11 V453
V10 G454

Figure 6. The docking pose of the ZIKV envelope solved structure (PDB ID: 5GZN) with GRP78 structure (PDB ID: 5E84) is achieved using the HADDOCK web server.
Colored cartoons represent the structures according to the domains. GRP78 nucleotide-binding domain (NBD) in dark green, GRP78 substrate-binding domain a
(SBDa) in yellow, GRP78 substrate-binding domainb(SBDb) in lime, ZIKV envelope domain III in sky blue and the rest of the envelope proteins in red. The active
amino acids involved in the interaction are represented in dark blue and yellow sticks for ZIKV envelope and GRP78, respectively.

Full-length ZIKV envelope - GRP78 binding
Before dealing with the docking data, a redocking experi-
ment is performed for the docking software to test whether
it is trustworthy for the present system under investigation
or not. The peptide binding structure of GRP78 (PDB ID:
5E84) is used to re-dock the peptide NR (a unique peptide
solved with Hsp70 structures that consist of 7 residues
NRLLLTG) to its binding site. Then the resulting docking pose
is compared with that of the solved structure (NR peptide is
part of the solved structure for crystallization procedures).
The RMSD between the NR peptide of the solved structure
and that docked using the HADDOCK web server is 1.51 Å.
Figure 6 shows the best model (based on the docking
score generated by the HADDOCK web server) for the dock-
ing of the ZIKV envelope structure (PDB ID: 5GZN) with
GRP78 solved structure (PDB ID: 5E84). The number of struc-
tures of the best docking score cluster is 160, with a docking
score value of 112.6 kcal/mol and a standard deviation of
15.8. As shown from the enlarged panel, the docking occurs
between the substrate-binding domain b of the GRP78 (lime
colored cartoon) and the domain III of ZIKV envelope protein
(sky blue colored cartoon). After analyzing the docking com-
plex, the residues involved in the interaction are I426, T428,
V429, V432, T434, F451, S452, V457, and I459 (yellow sticks)
from GRP78 and C308, T309 and A311 (blue sticks) from ZIKV
envelope protein. The interactions are established and main-
tained by mainly the hydrophobic residues (C308, T309, and
A311) that lie in the predicted region of ZIKV envelope pro-
tein near the disulfide bond. The same conclusion can be
reflected from Table 2, where the different dynamical states
(during 50 ns MDS run) of the ZIKV Envelope protein is
tested against GRP78. The results agree with that of the
HpepDOCK in the previous section. This further supports our
hypothesis of the binding site, as shown in Figure 6.
The predicted binding site for ZIKV envelope protein to
GRP78 in this study is in good agreement with previous
studies that proved the recognition of ZIKV envelope domain
10 A. A. ELFIKY AND I. M. IBRAHIM

Table 2. The binding affinity pattern of the docking of GRP78 (PDB ID: 5E84) with the different conformations of ZIKV full-length Envelope protein (at 10.9,
17.1, 20.4, 28.5, 33.9, 34.8, 38.1, 43.4, and 44.5 ns). Bold residues are the same residues reported in other docking trials of HpepDock and HADDOCK. Red-colored
residues are salt bridges, while the blue-colored residue is p-cation interaction.
H-bonding Hydrophobic interaction
Residues
ZIKV Envelope HADDOCK Residues from Residues from ZIKV Residues
Conformation at score number ZIKV Envelope from GRP78 number Envelope from GRP78
10.9 ns 70.3 5 C308 T434 10 Y305 V453
± 2.7 Q344 K447 L307 T428
N362 V490 L307 T434
N362 Q492 L307 F451
G392 K435 L307 F451
T309 T428
T309 V432
D336 V429
L352 K447
N362 V490
17.1 ns 85.5 10 F312 G454 7 A311 V453
± 0.8 Q331 G489 T313 V490
Q350 K460 A333 V490
Q350 D525 V391 V429
N371 G489 E393 T428
E393 T428 E393 I459
E393 T428 I396 V453
E393 T428
K395 T458
K395 T458
20.4 ns 63.3 10 T309 T428 8 T309 V432
± 2.5 T309 T434 A311 F451
F312 S452 F314 V490
T313 S452 F314 V490
T313 Q492 V391 V429
A333 Q449 K394 T428
T335 K435 K394 F451
E393 T458 W400 R488
W400 R488
E370 K447
28.5 ns 60.7 7 T309 V429 5 T309 V432
± 2.2 F312 S452 A311 F451
T313 Q492 F314 V490
F314 S452 V391 V429
E370 Q449 E393 V457
K394 E427
E370 K447
33.9 ns 83.3 17 T309 V429 7 T309 V432
± 3.0 F312 S452 A311 T428
F314 S452 T313 I450
F314 Q492 F314 V490
P318 R488 F314 V490
P318 R488 V391 V429
T335 T434 K394 T428
T335 K435
E370 Q449
E393 E427
E393 T428
H398 R488
H398 G489
H399 A486
H399 G489
E370 K447
E393 K460
34.8 ns 57.0 11 L307 T434 5 T309 I426
± 0.9 C308 T428 T309 T434
T309 T434 T313 V490
T309 Q449 E370 I450
A311 S452 V391 V429
T335 S448
T335 Q449
T335 Q449
T335 I450
D336 Q449
E370 K512
38.1 ns 64.7 8 T309 V429 7 T309 V432
± 4.4 F314 S452 A311 F451
E393 T428 F314 V490
(continued)
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 11

Table 2. Continued.
H-bonding Hydrophobic interaction
Residues
ZIKV Envelope HADDOCK Residues from Residues from ZIKV Residues
Conformation at score number ZIKV Envelope from GRP78 number Envelope from GRP78
E393 K460 F314 V490
T397 G454 V391 V429
W400 R488 K394 T428
W400 R488 K394 F451
E370 K447
43.4 ns 54.4 7 S306 V429 6 T309 F451
± 5.8 T335 T434 T335 K435
T335 K435 D336 T428
D336 T434 L352 V490
S368 K435 E393 I450
G392 S452 K394 I450
E370 K447
44.5 ns 74 14 V303 S452 7 Y305 T428
± 1.5 V303 S452 Y305 V429
Y305 V429 Y305 V432
L307 I450 L307 I450
C308 Q449 T335 K435
T309 S448 T335 P438
T309 Q449 V391 I450
T335 K435
D336 T434
D336 Q449
G337 K435
P338 T434
S368 K435
D336 K447

III by neutralizing antibodies (Pujhari et al., 2017). This bind- Author contribution
ing site would open the door for in depth experimental stud-
AAE; own the research idea, perform MDS calculations, and wrote the
ies and also simulations on the mode of envelope protein manuscript. IMI; did the docking calculations and revised
determination by the GRP78 substrate-binding domain. the manuscript.

Conclusion Data availability

ZIKV envelope protein is an essential viral element that helps
in the internalization of virus particles to the host cell.
Several host cell receptors are the target of viruses, including
the cell-surface GRP78, which is overexpressed under stress.
Inhibiting the interaction that occurs between the viral enve-
lope protein and the host cell receptor would probably
decrease the rate of viral infection. Besides, a vaccine against
ZIKV envelope protein would likely prevent viral infection.
The present computational study suggests a ZIKV envelope
protein - GRP78 binding site, thus paving the way for drug
designers to develop suitable inhibitors to prevent the bind-
ing and hence the infection. Future work involving the
dynamics of GRP78 and experimental validation would be
required to be able to suggest potent peptidomi-
metic inhibitors.
Acknowledgements
MDS was performed on the Bibliotheca Alexandrina supercomputing
facility, Alexandria, Egypt. We want to acknowledge the help of Prof. Dr.
Wael Elshemey and Mrs. Alaa Ismail for the helpful discussion and revi-
sion of the manuscript.
Disclosure statement
No potential conflict of interest was reported by the author(s).
The docking structures are available upon request from the correspond-
ing author
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