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\begin{document}

\title{Conformational changes in the AdeB transmembrane efflux pump by
amphiphilic peptide Mastoparan-B, down-regulates expression of the ade B
gene and restores antibiotics susceptibility}



\author[1]{Mohammad Reza Shakibaie}%
\author[2]{Farzan Modaresi}%
\author[3]{Omid Azizi}%
\author[1]{Omid Tadjrobehkar}%
\author[1]{Mohammad Mehdi Ghaemi}%
\affil[1]{Kerman University of Medical Sciences}%
\affil[2]{Jahrom University of Medical Science}%
\affil[3]{Torbat Heydarieh University of Medical Sciences}%


\vspace{-1em}



  
  \date{May 28, 2024}


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\selectlanguage{english}
\begin{abstract}
No report exists on the role of Mastoparan B (MP-B) as an RND efflux
pump inhibitor in multi-drug resistant (MDR) \emph{Acinetobacter
baumannii}. Here, we performed a series of in-silico experiments to
predict the inhibition of the AdeB efflux pump by MP-B as a drug target
agent. For this reason, an MDR strain of \emph{A. baumannii} was
subjected to minimum inhibitory concentration (MIC) against 12
antibiotics as well as MP-B. Expression of the a \emph{de}B gene in the
absence and presence of sub-MIC of MP-B was studied by qRT-PCR. It was
found that MP-B had potent antimicrobial activity (MIC=1 \selectlanguage{greek}µ\selectlanguage{english}g/ml)
associated with a 20-fold decrease in its expression at sub-MIC of MP-B.
The stereochemical analysis using several automated servers confirmed
that the AdeB is an inner membrane of the RND tripartite complex system
with helix-turn-helix conformation and a pore rich in Phe, Ala, and Lys
residue. The best model that showed high accuracy (Z=1.2, C=1.41,
TM=0.99, and RMSD=4.4) was selected for docking purposes using the Site
Map tool, and the correct protein-peptide complexes were simulated in
the BioLiP platform. The molecular docking via AutoDock/Vina suggested
that MP-B form H-bound with amino acid residues of the AdeB helix-5 and
caused a shift in the dihedral angle by distances of 9.0 \selectlanguage{ngerman}Å, 9.3 Å, and
9.6 Å, respectively. This shift was detected by the AlphaFold 2 tool and
influenced the overall druggability of the protein. From the results, we
concluded that, MP-B can be a good candidate for inhibition of bacterial
efflux pump.%
\end{abstract}\selectlanguage{ngerman}%



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\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

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\end{figure}

\textbf{1 INTRODUCTION}

Computation chemistry has greatly increased our knowledge about protein
conformation and drug interaction and helped us better understand
microbial infection treatment.\textsuperscript{1} Biomolecular
simulation with models allows for investigations of target proteins'
structure and function features, which are useful for identifying
drug-binding sites and elucidating drug action
mechanisms.\textsuperscript{2,3} Recently a breakthrough in the 3D
construction of various proteins occurred with the invention of the
AlphaFold Protein Structure Database in cooperation with EMBL-EBI.
AlphaFold can accurately predict 3D models of protein structures and is
accelerating research in nearly every field of biology such as
drug--target interaction.\textsuperscript{4}

In this regard, \emph{A. baumannii} is a Gram-negative hospital-acquired
pathogen to blame for high morbidity and mortality within the medical
care units such as ICUs of various hospitals across the
world.\textsuperscript{5} Following the increasing emergence of strains
that are not susceptible to any clinically used antibiotics, the WHO has
ranked carbapenem-resistant strains of this bacterium in first place in
its global priority pathogen list.\textsuperscript{6} \emph{A.
baumannii} has different resistance mechanisms against antimicrobial
agents such as \selectlanguage{greek}β-\selectlanguage{english}Lactam inactivation, biofilm formation, efflux pumps,
alterations of the affinity for antibiotics,
etc.\textsuperscript{7-9}One of the most important mechanism of
resistance to antibiotics is the efflux pump which plays a key role in
the intrinsic resistance to several antibiotics.\textsuperscript{10,11}
The efflux pumps are divided into five superfamilies: ATP-binding
cassette (ABC), small multi-drug resistance (SMR), multi-antimicrobial
extrusion (MATE), major facilitator (MFS), and
resistance/nodulation/division (RND).\textsuperscript{12} The
best-characterized multi-drug efflux system in \emph{A. baumannii} is
the RND superfamily.\textsuperscript{13} These tripartite complexes
comprise an inner membrane that acts as a secondary active
H\textsuperscript{+}/drug antiporter (AdeB), extruding a vast spectrum
of structurally unrelated drugs through a periplasmic membrane fusion
protein channel (AdeA), connected to an outer membrane funnel shape
channel (AdeC).\textsuperscript{14} The primary RND efflux pump of
\emph{A. baumannii} (AdeABC) was first described in MDR strain
BM4454.\textsuperscript{14} The \emph{Ade} abc locus has consisted of
three tandemly linked genes encoding AdeA, AdeB, and AdeC proteins and
showed resistance to several antibiotic classes such as aminoglycosides,
tetracyclines, and fluoroquinolones,
respectively.\textsuperscript{15}The AdeB is an inner membrane protein
and transports various drugs through the central
pore.\textsuperscript{16} The pore is opened to the periplasm via three
vestibules located at subunit interfaces and a central
cavity.\textsuperscript{17} More researchers demonstrated that, the
\emph{ade} B is the most important gene in the adeABC efflux
system.\textsuperscript{17,18} Genetic studies suggested that
discrimination between the substrates in \emph{A. baumannii} occurs
mainly within the periplasmic region instead of the transmembrane
region.\textsuperscript{19} Cryo-Electron Microscopy (CEM) of the
monomers of the inner membrane protein suggested that AdeB can adopt
three different states: access (loose), binding (tight), and extrude
(release) to provide essential dynamics for the efflux
process.\textsuperscript{20} The AdeB trimer primarily adopts mainly a
resting state (absence of antibiotics) with all promoters in a
conformation devoid of transport channels or antibiotic binding
sites.\textsuperscript{21}

Transmembrane proteins are relevant for drug development since they make
up more than 50\% of all human drug targets given that approximately
25\% of all the proteins.\textsuperscript{22} Membrane proteins are
difficult to study due to their partially hydrophobic surfaces,
flexibility, and lack of stability.\textsuperscript{21} Consequently
creates the need for bioinformatics tools that can accurately identify
these types of proteins by predicting their topology, position, and
orientation as well as their N-terminal utilizing the contact map with
coevolutionary precision matrices.\textsuperscript{23} Several methods
have been developed over the last decades that predict protein topology
with high accuracy. Many of these methods are freely available as web
servers, both individually or combine with several other bioinformatics
tools.\textsuperscript{24}

In the past 20 years, scientists were unable to produce effective
antibiotics against Gram-positive or Gram-negative pathogens.
Furthermore, the use of nanoparticles, gene therapy, phage therapy, and
immunotherapy are either costly or still in their
infancy.\textsuperscript{25} In contrast, the amphiphilic antimicrobial
peptides like Mastoparan B (a cationic, tetradecaeptide
LKLKSIVSWAKKVL-CONH2 isolated from the venom of the hornet Vespa
basalis) at low concentrations has potent antimicrobial activity and can
be used along with antibiotics for treatment of various
diseases.\textsuperscript{26} These compounds frequently destabilize
biological membranes by dual amphiphilic activity resulting in membrane
perforation.\textsuperscript{27} In recent years, numerous efflux pump
inhibitors have been discovered and tested, including natural products,
antibiotics, and synthetic molecules.\textsuperscript{28} The
combination of antibiotics with natural products circumvents the
resistance phenomenon and consequently decreases the dose and drug side
effects. The activity of these peptides in the combination with
antibiotics on \emph{Escherichia coli} AcrAB-TolC and
the\emph{Pseudomonas aeruginosa} MexAB-OprM complexes were well
delineated.\textsuperscript{29} In contrast, there is no information
about the effect of Mastoparan-B on the RND efflux in \emph{A.
baumannii} . It has been reported that the phenylalanine-arginine
\selectlanguage{greek}β-\selectlanguage{english}naphthylamide (PA\selectlanguage{greek}β\selectlanguage{english}N) a broad-spectrum efflux pump inhibitor interacts
with AdeB strongly through the distal binding
pocket.\textsuperscript{30} In another investigation, computational
screening suggested that Strictamin (Akuammilan-17-oic acid methyl ester
from Alstonia scholaris) and Limonin (7, 16-dioxo-7, 16-dideoxylimondiol
from Citrus spp.) exhibited binding to RND efflux pump in MDR strains
of\emph{A. baumannii} .\textsuperscript{31}

Therefore, in the present study, for the first time, we are reporting
transcriptional analysis of the \emph{ade} B gene in the presence of
Mastoparan-B, the detailed stereochemical structure of AdeB protein
before and after binding to this peptide, superfamily analysis by CATH
database, Gene Ontology, molecular docking, prediction of structure
conformation and helix folding by AlphaFold Protein Structure Database
(AlphaFold 2). The emerging picture using this peptide in combination
with antibiotics further suggests opportunities for drug designs.

\textbf{2 MATERIALS AND METHODS}

2.1 Bacterial source and PCR-Sequencing

An MDR strain of \emph{A. baumannii} was isolated from the previous
collection \textsuperscript{32} and used for the detection of
the\emph{ade} B gene by polymerase chain reaction (PCR) technique.
Briefly, total genomic DNA was extracted by a DNA genomic extraction kit
(ParsTous, Iran). The amplification was carried out using a common PCR
core mixture (total volume, 50 \selectlanguage{greek}µ\selectlanguage{english}l) containing 20 \selectlanguage{greek}µ\selectlanguage{english}L PCR buffer, 10 \selectlanguage{greek}µ\selectlanguage{english}L of
200 mM each dNTP (Bioneer, South Korea), 1 U of AmpliTaq DNA polymerase
(ParsTous, Iran), 20 pmol of the forward -5' TTAACGATAGCGTTGTAACC -3'
and reverse 5'- TGAGCAGACAATGGAATAGT -3' primer pair (Bioneer, Korea)
and 50 ng of the genomic DNA as the PCR template. The \emph{ade} B gene
was further purified by a DNA purification kit (ParsTous, Iran) and
sequenced in both directions by Sanger's dideoxy chain termination
method using ABI Prism 373 DNA Sequencer (Applied Biosystems 373/3730XL,
Bioneer, Korea). The \emph{ade} B sequence was then submitted to the
GenBank NCBI database (https://www.ncbi.nlm.nih.gov/genbank/submit/) for
accession number. The BLAST program was used to find the amino acid
sequence of the AdeB protein.

2. 2 Susceptibility to antibiotics and Mastoparan-B

The susceptibility of the selected strain to 12 antibiotics was carried
out by disc diffusion assay and MIC methods using broth microdilution
test by the Clinical and Laboratory Standards Institute (CLSI 2019)
protocol.\textsuperscript{33} At the same time, the susceptibility of
the isolate to Mastoparan-B was performed by microdilution method using
the Tryptic Soy broth medium, and criteria for sensitivity or resistance
were measured based on our previous
report.\textsuperscript{34}Similarly, we performed MICs of antibiotics
in the presence of sub-MIC concentration (0.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL) of MP-B to see any
synergism between peptide and antibiotics. Before the experiment, we
checked the viability of the cells at this concentration, by removing 1
ml aliquots of each sample grown in the presence and absence of 0.5 ml
of MP-B at each 2 h and reading the turbidity with a UV/Vis
spectrophotometer (Persia Med, AE-S60-4U) at an optical density (OD) of
600 nm. The standard cultures of \emph{E. coli} ATTC25922 and \emph{A.
baumannii} ATCC 19606 were used as the quality control strains. All
antibiotics were purchased from MAST Company, Manchester, UK.
Mastoparan-B powder with 99.5\% purity was purchased from Shimi-Daru
(Tehran, Iran) and used as described by the manufacturer.

2. 3 Expressional analyses of the \emph{ade} B Gene

RNA extraction, C-DNA synthesis, and relative quantitative real-time PCR
(qRT-PCR) were performed to assess the expression of the \emph{ade} B
gene in the presence and absence of 0.5 \selectlanguage{greek}µ\selectlanguage{english}g/ml of MP-B as described
previously.\textsuperscript{32} Briefly, amplification was done using 25
\selectlanguage{greek}µ\selectlanguage{english}l of SYBR\selectlanguage{ngerman}®Green dye and 2× Real-Time PCR master mix solution (BIO FACT,
Korea), followed by the addition of 1 \selectlanguage{greek}µ\selectlanguage{english}l of \emph{ade} B primer pair F:
5'-\selectlanguage{english}AACGGACGACCATCTTTGAGTATT-3' \selectlanguage{english}and R: 5- '\selectlanguage{english}CAGTTGTTCCATTTCACGCATT-3' \selectlanguage{english}and
5 \selectlanguage{greek}µ\selectlanguage{english}l of cDNA template. Quantification of the \emph{ade} B gene was
performed by using the ABI Step One qRT-PCR system (Applied Biosystems,
Foster City, CA, USA). The gene expression was calculated as a fold
change (RQ) between the target gene and matched reference 16SrRNA levels
based on the following formula RQ = \selectlanguage{greek}\textsuperscript{2Δ}\selectlanguage{english}DDCt. Where \selectlanguage{greek}Δ\selectlanguage{english}Ct
is equal to the difference between the cutting point (Ct) value for the
analyzed gene and Ct for the 16SrRNA reference gene as described by
Livak et al.\textsuperscript{35} \emph{A. baumannii} ATCC 19660 was used
as the internal control strain. Differences were assessed by Student's
t-test for consideration as statistically significant.

2. 4 Classification, physicochemical parameters, and Gene Ontology

The hierarchical classification of AdeB protein to the superfamily,
homologous family, predicting domains, and important functional sites
were carried out by the InterPro software suite
(https://www.ebi.ac.uk/interpro/). To classify proteins in this way,
InterPro uses predictive models, known as signatures provided by several
different alignments and produces a comprehensive resource for protein
classification (https://www.ebi.ac.uk/interpro/search/sequence/). Any
alignment with a TM-score [?]0.5, which is the cutoff value for a
significant structural match, was used as a potential template for the
target protein-peptide interaction. Furthermore, molecular weight,
theoretical pI, amino acid composition, atomic composition, extinction
coefficient, estimated half-life, instability index, aliphatic index,
and grand average of hydropathicity (GRAVY) were computed by information
stored in the Swiss-Port ProtParam database
(https://web.expasy.org/protparam/). Gene Ontology at molecular and
cellular functions was determined using the UniProtKB platform
(https://www.uniprot.org/). We also worked on the domain-based method
for the AdeB protein sequence versus structural diversity and prediction
of the functional site that exploits functional sub-classification of
the superfamilies by the CATH protein classification database
(https://www.cathdb.info/).

2. 5 Construction of phylogenetic tree of the AdeB protein

The phylogenetic tree of the AdeB protein was constructed using maximum
likelihood sequence alignment Clustral Omega version 7 in the UniProtKB
pipeline, and variable sites were removed by the stringent
settings.\textsuperscript{36} A bootstrapped was generated with RAxML
(Randomized Accelerated Maximum Likelihood) version~7.0.3 with the
confidence levels (\%) generated from 1000 bootstrap
trials.\textsuperscript{36} Moreover, we choose a consensus sequence
manually using similar protein sequences from the UniProtKB restricted
to the \emph{A. baumannii} AdeB protein.

2. 6 Protein dynamic simulation

From the primary amino acid sequence, the 3D model of AdeB protein was
generated by the SWISS-Expasy (https://swissmodel.expasy.org), and
I-TESSAR (https://zhanggroup.org/I-TASSER/) platforms using the homology
modeling program. For each target, I-TASSER simulation generates a large
ensemble of structural conformations, called decoys. To select the final
models, I-TASSER uses the SPICKER program to cluster all the decoys
based on the pair-wise structural similarity and reports up the top five
models which correspond to the five closest structure clusters. The
confidence of each model is quantitatively measured by the C-score that
is calculated based on the significance of threading template alignments
and the convergence parameters of the structure assembly simulations.
Furthermore, for the evaluation of multiple, amino acids alignments and
similarity matrix, we used the FASTA format of the targeted amino acids
sequence and blasted in the UniProtKB database
(https://www.uniprot.org/). The functional domains of the AdeB protein
were bolded using the ROSETTA prediction tool
(https://www.rosettacommons.org/) and the Hidden Markov Model program
(HMMs) (https://www.ebi.ac.uk/Tools/hmmer/). The quality of obtained
models was validated with a comprehensive scoring function for model
quality assessment servers such as ProSA and QMEAN.\textsuperscript{38,
39} The dihedral angles including phi (\selectlanguage{greek}φ\selectlanguage{english}) and psi (\selectlanguage{greek}ψ\selectlanguage{english}) and backbone
conformation were assessed using the PROCHECK tool at the PDB Sum server
(https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/). In the field of
drug discovery, the use of 3D models can provide an understanding of why
a certain drug compound is an inhibitor, while a related compound is
not, or why certain proteins are druggable while others are not.
Recently, the AlphaFold DB database resolves this problem and showed
folding of protein plays a role in the druggability of targeted protein.
In this study, we evaluated the accuracy of the AdeB protein structure,
folding patterns of the helices and domain side chains including the
interior side of the AdeB by the AlphaFold 2 database
(https://alphafold.ebi.ac.uk). In addition, we computed the backbone
dihedral angles (F\selectlanguage{english}) to investigate rotameric conformational for the
amino acid residues binding with ligand base on the Ramachandran plot
and two other model simulation apo and ligands.\textsuperscript{40}

2. 7 Prediction of the protein-peptide interaction site

Because of peptide flexibility and the transient nature of
protein-peptide interactions, peptides are difficult to study
experimentally. Therefore, we used computational methods for predicting
structural information about protein-peptide interactions by the
InterPep server (http://wallnerlab.org/InterPep/). The InterPep software
is a powerful tool for identifying peptide-binding sites with a
precision of 80\% at a recall, of 20\%. It is an excellent starting
point for docking protocols or experiments investigating peptide
interactions.

2. 8. Ligand preparation and molecular docking

Mastoparan-B with MF: C78H138N20O16 was retrieved from the PubChem
database (Compound CID: 86289587) and used as a ligand in this study. 20
different ligand poses were selected using the LigPrep module in Maestro
v11 employing the Schr\selectlanguage{ngerman}ödinger platform.\textsuperscript{41} The best
interaction poses were visualized using the PyMOL server
(https://pymol.org). The most comprehensive and accurate model of
ligand-protein docking and the protein function annotation was obtained
from the BioLiP database (http://zhanglab.ccmb.med.umich.edu/BioLiP/).
The free energy was minimized by the Universal Force Field and converted
to the pdbqt format in PyRx0.8 for virtual
screening.\textsuperscript{42} Furthermore, the favorable potential drug
target interactions between the selected ligand molecule and modeled
receptor (AdeB) were identified by the extra precision (XP) feature of
Grid-based Ligand Docking with Energetics (GLIDE) in the Schrödinger
platform.\textsuperscript{42} Each simulation was repeated three times
with different initial conditions to increase the precision of the
simulations and to prevent any dependencies of the results on the
initial conditions. Binding sites were generated using the Site Map tool
version 2.4 (Schrödinger Inc.). Finally, molecular docking was performed
via AutoDock/Vina suite to assign the ligands and receptors bond orders
by adding hydrogen atoms.\textsuperscript{43} For docking purposes, the
best docking pose was selected directly using the Glide G-Score as
described previously.\textsuperscript{41} Once G-Score was obtained, it
was used to build optimal binding free energy that distinguishes our
sequence from the reference sequence (usually taken to be the optimal
sequence). Site Map was tasked to identify the five top-ranked possible
receptor suites using the default settings which include size, volume,
amino acid exposure, enclosure, contact, hydrophobicity, hydrophilicity,
and donor/acceptor ratio. Furthermore, the topology of the
transmembrane, inside and outside \selectlanguage{greek}α-\selectlanguage{english}helices was predicted by the
DeepTMHMM (https://dtu.biolib.com/DeepTMHMM) and TOPCONS
(http://topcons.net/) modules.

2. 9 Preparation of the contact map and domain boundaries

We predicted the AdeB protein contact map and domain boundaries using
deep residual neural networks coupled with coevolutionary precision
matrices (https://zhanglab.ccmb.med.umich.edu/FUpred). A protein contact
map represents the distance between all possible amino acid residue
pairs of a 3D structure. For two residues, the element of the matrix is
1 if the two residues are closer than a predetermined threshold, and 0
otherwise.\textsuperscript{44} To achieve reliable results, we used the
FUpred score for a continuous two-domain protein, using the following
formula

FUscore2\emph{c} (\emph{l} ) =2\emph{N} 1, 2(\emph{l} ){[}1\emph{N}
1(\emph{l} )+1\emph{N} 2(\emph{l} ){]}

where \emph{l} is the domain splitting point of a protein,\emph{N}
\textsubscript{1} (\emph{l} ) and \emph{N} \textsubscript{2}(\emph{l} )
represent the number of contacts within the first and second domains,
respectively, and \emph{N} \textsubscript{1, 2}(\emph{l} ) =\emph{N}
\textsubscript{2, 1}(\emph{l} ) indicates the number of contacts between
the first and second domains.\textsuperscript{45} The core idea of the
algorithm was to retrieve domains boundaries locations by maximizing the
number of intradomain contacts while minimizing the number of
interdomain contacts from the contact map.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

2. 10 Statistics

Statistical analyses were performed using SPSS 17.0 (SPSS, Chicago, IL,
USA). A p [?] 0.05 was considered statistically significant for two-tailed
tests.

\textbf{3 RESULTS}

3.1 Bacterial source and identification

In our previous reports, 65 isolates of \emph{A. baumannii} were
isolated from the ICUs hospitals in Kerman,
Iran.\textsuperscript{31}Fifty-five of these were MDR, while 10 were
carbapenems susceptible. Among them, one isolate that exhibited the
highest MICs to various classes of antibiotics was selected for this
study.

3.2 Antimicrobial susceptibility tests and PCR-Sequencing

The organism exhibited high MICs against gentamicin and kanamycin (64
\selectlanguage{greek}µ\selectlanguage{english}g/ml), ciprofloxacin and levofloxacin (125 \selectlanguage{greek}µ\selectlanguage{english}g/ml), ceftazidime and
ceftriaxone (256 \selectlanguage{greek}µ\selectlanguage{english}g/ml), imipenem (125 \selectlanguage{greek}µ\selectlanguage{english}g/ml), colistin (8 \selectlanguage{greek}µ\selectlanguage{english}g/ml),
piperacillin/tazobactam (125 \selectlanguage{greek}µ\selectlanguage{english}g/ml), and azithromycin (64 \selectlanguage{greek}µ\selectlanguage{english}g/ml),
respectively (Figure. 1A). Furthermore, MP-B prevented the active growth
of the MDR strain of \emph{A. baumannii} with mean MIC=1 \selectlanguage{greek}µ\selectlanguage{english}g/ml, where
99.9\% of the cell's population was not grown in Tryptic Soy broth
medium (p[?]0.05). A significant synergy was observed when the cells were
grown in the presence of both antibiotics (various concentrations) and
sub-MIC of MP-B (Figure 1A). The MICs against gentamicin and kanamycin
were reduced to 8 \selectlanguage{greek}µ\selectlanguage{english}g/ml, ciprofloxacin and levofloxacin to 4 \selectlanguage{greek}µ\selectlanguage{english}g/ml,
ceftriaxone to 2 \selectlanguage{greek}µ\selectlanguage{english}g/ml, carbapenems to 4 \selectlanguage{greek}µ\selectlanguage{english}g/ml, and tetracycline to 2
\selectlanguage{greek}µ\selectlanguage{english}g/ml, respectively. However, no change in the MIC level of colistin,
piperacillin-tazobactam, azithromycin, co-trimoxazole, and
chloramphenicol was observed (Figure. 1A). To confirm the synergism of
Mastoparan-B with antibiotics, we performed growth curve analysis of the
isolate in the presence and absence of sub-MIC of MP-B. When cells grown
in 0.5 \selectlanguage{greek}µ\selectlanguage{english}g/ml of MP-B, relatively steady growth was observed after 8
hours of incubation (Figure. 1B). The results indicated that the
reduction in MIC against antibiotics was not due to the death of
bacteria by Mastoparan-B but rather the inactivation of some cellular
mechanism (s) such as efflux pump.

To prove this hypothesis, we performed the PCR technique by using
specific primer pairs for the entire \emph{ade} B gene. The AdeB efflux
pump gene was then sequenced using Sanger's dideoxy DNA chain
termination method by ABI Prism DNA Sequencer, and its expression was
evaluated in the presence and absence of MP-B. Interestingly, a 20-fold
decrease in the expression of the \emph{ade} B efflux pump gene was
detected when the cells were grown at the sub-MIC level of this peptide.
Furthermore, we obtained the nucleotide sequence of the \emph{ade} B
gene by blasting with similar sequences in the NCBI database
(https://www.ncbi.nlm.nih.gov/blast). From the nucleotides, we detected
the AdeB protein sequence in the GenBank database.

3.3 Physicochemical parameters and Gene Ontology

The computation of physicochemical parameters of the AdeB efflux pump
revealed that the protein was typically composed of 1036 amino acid
residues with an average molecular weight (M. Wt.) of 112592.02 and the
molar extinction coefficient of (\selectlanguage{greek}ε\selectlanguage{english}) 81945 (Table 1A). Likewise,
empirical analysis of amino acid composition by the Expasy ProtParam
database revealed the dominance of small aliphatic amino acids like
alanine at 16\%, isoleucine, valine at 10\%, and glycine at 6.7\% in the
AdeB structure (Table 1B). Furthermore, documentation resources of
protein families, active domains, and functional sites indicated the
AdeB protein belonged to the RND transporter efflux (HAE1) similar to
ArcB protein Acrflvin-R, and ACR\_tran family. It showed the
nonhomologous relationship with superfamily ArcB\_ DN\_DC and
pore-TolC-like domains. Gene Ontology further confirmed the biological,
and functional activities of the AdeB protein. The overview of the
sequence/structure diversity of the RND superfamily in this study
compared to other superfamilies in the CATH database is presented in
Figure. 2A. The obtained functional and structural domains by CATH were
similar to MDR efflux transporter ArcB transmembrane domains with two
related sequence families and one structural cluster (Figure 2A). The
AdeB efflux protein had 91.8\% unique sequence annotations (Figure 2B)
and, the lineage had 6 functional family members in the genus
Acinetobacter (Figure 2C).

3.4 Phylogenetic tree analysis and amino acids alignments

The phylogenetic tree analysis of the AdeB protein convincingly
demonstrated high sequence identity (99.9\%) with AdeB efflux pump
membrane transporter tr-AOA7L9E4V2\_ACIBA, tr-A0A5P6FTF2\_ACIBA, and
tr\textbf{-} A0A3Q8ULEO\_ACIBA, 99.6\% sequence identity with
tr-AOA373B8P5\_9GAMM, and 99.41\% identity with efflux pump
tr-A0A086HUIO\_ACIBA, respectively (Figure 3A). Moreover, we also
investigated blast parameters including, identity (99\%), score (Average
50300), and an E-value (5.3-11) which indicated significant accuracy in
clustral pattern (Figure 3B). To prove the above results, we performed
multiple amino acid alignments of the AdeB and compared the amino acids
helix-5 for closely related strains, the results revealed high identity
among the amino acids of these strains (Figure 3C). Overall, the high
identity matrix of the AdeB protein sequence was observed in the
UniProtKB database ([?]99\%) (Figure 3D). This indicates that a high
evolutionary relationship exists among sequences in this report.

3.5 Prediction of the 3D structure of AdeB protein

The obtained AdeB trajectory showed an inner membrane protein with a
3-fold asymmetrical axis positioned perpendicular to the membrane
surface with the typical RND-like folds. The predicted RND efflux pump
exhibited three components; i) a 30\selectlanguage{ngerman}Å U shape funnel channel in the AdeC
outer membrane region composed of both \selectlanguage{greek}α- \selectlanguage{english}helices and \selectlanguage{greek}β-\selectlanguage{english}sheets (Figure
4A), ii) a 40 \selectlanguage{ngerman}Å large periplasmic membrane fusion AdeA region with the
coil-coil conformation, and iii) a 50Å transmembrane AdeB transporter
helical structure in the form of helix-turn-helix with a pore (Figure
4A). The large periplasmic domain of the AdeB molecule is created by two
extracellular loops that link together TM1 with TM2 and TM7 with TM8.
This periplasmic domain can be divided into six subdomains, PN1, PN2,
PC1, PC2, DN, and DC (Figure 4B). These features fit with the extreme
variety of antibiotics recognized by AdeB. The N-terminal TM1 leads to
the PN1 subdomain that connects the PN2. Similarly, TM7 leads to the PC1
subdomain that was connected to the PC2 (Figure 4B). Nevertheless, a
pore connected the inner membrane cleft to the periplasmic cavity
(Figure 4C). The amino acids analysis of the pore displayed a high
quantity of Lys, Phe, and Ala residues (Ala residue relatively
stabilized the pore channel) (Figure 4C). The combination of deep neural
network learning with completed I-TASSER assembly significantly improved
by threading template quality and therefore boosted the accuracy of the
final model through optimized fragment assembly simulations with C-score
= 1.41, TM- score =0.99, RMSD (Å) = 4.4 ± 2.9, and P-score = 1,
respectively. Nevertheless, the validity of the AdeB structure was
further confirmed by the Ramachandran plot (Figure 5A) and local
similarity estimate of amino acid residues in chains A, B, and C (Figure
5B). The results indicated that over 99\% of the amino acid backbones
remained in the Ramachandran favored regions and less in the outlier.
The plot also illustrated torsional angles at low clash point \selectlanguage{greek}ψ \selectlanguage{english}(0.87\%)
and high Ramachandran favored F \selectlanguage{english}(97.92\%) conditions. The obtained
results of the local quality estimate by the Swiss-Expasy database for
all three chains illustrated consistency in our work. Furthermore, the
structural validation of the secondary protein structure using the PROSA
website (Figure 5C) showed the generated model has a Z-score = 2.3,
which is within the acceptable range of -10 to 10.

3.6 Molecular docking analysis

For docking of Mastoparan-B with the AdeB protein, we prepared 20 poses
and used them to determine the suitable binding site on the AdeB efflux
pump. The comparison of the molecular docking simulation before and
after attachment of MP-B showed MP-B attached exclusively with the
helix-5 inner membrane via Val 499, Phe 454, Thr 474, Ser 461, Gly 465,
and Tyr 468 residues, respectively (Figure 6A). Upon docking of the
ligand, there was a change in energy minimization (glide score of 0.8 to
0.2), and a shift in the orientation of dihedral angles including phi
(\selectlanguage{greek}φ\selectlanguage{english}), and psi (\selectlanguage{greek}ψ\selectlanguage{english}) by distances of 9.0 \selectlanguage{ngerman}Å, 9.3 Å, and 9.6 Å (Figure 6B).
The black arrows indicate the bond interaction potentials:
bond-stretching, VB, angle-bending, VA, dihedral (out-of-plane), Vdh.
The protein folding changes were detected by AlphaFold DB software.
Here, we used the Alfafold database for the first time for the
determination of the AdeB domains and detailed information about folding
and the position of helix-5 in the interior side of the AdeB protein
(Figure 7A). In addition, the attachment of MP-B to this helix and amino
acids is involved in molecular docking studied by this software
represented in Figure 7B. Finally, the overall docking process, the
positions of the amino acids, and H-bonds with the ligand molecule are
shown in Figure 7C. Moreover, comparing the AdeB molecule with template
7cz9A as the model indicated a high score in the ligand-receptor
interaction (z-score = 33.510 cutoff = 6.9).

3.5 Prediction of the position and topology of transmembrane helices

The topology of AdeB protein was predicted by both DeepTMHMM and TOPCON
tools using Z-coordinates and predicted \selectlanguage{greek}Δ\selectlanguage{english}G-values across the sequence
and the Wilcoxon rank-sum test to get a quantified comparison between
the two reliability scores independent of the overall prediction
accuracy. The majority of transmembrane helices were arranged at
positions between 300-500 and 900-1000. The place of binding ligand to
the receptor is shown by a circle with a Z score of 5.8 (Figure 8A).
Furthermore, analysis by TOPOCONS further confirmed the above results
(Figure 8B), and estimated the percent of \selectlanguage{greek}α-\selectlanguage{english}helix inside, membrane, and
outside of the AdeB protein (Figure 8C). The transmembrane helices
(TMHs) topology constituted the highest percentage in the AdeB
structure. Furthermore, employing Pyr\textsuperscript{2} software we
determined 47.7\% of amino acid residues displayed \selectlanguage{greek}α-\selectlanguage{english}helical structure,
21\% \selectlanguage{greek}β- \selectlanguage{english}sheets, 11.5\% turns, and 24.5\% coil with the Z score +2.52. As
a result, the most frequent topology in AdeB protein was \selectlanguage{greek}α-\selectlanguage{english}helix,
followed by a random coil and extended \selectlanguage{greek}β-\selectlanguage{english}strand barrel.

3.8 Contact map of AdeB protein

A protein contact map represents the distance between all possible amino
acid residue pairs of a three-dimensional protein structure. Here we
carried out the contact map analysis of AdeB transporter by using the
FUpred server. There were three overlapping D1-3, D6-2 and D6-1 at the
N-terminal segment with a confidence score of 8.76 (Figure 9A). The
contact maps derived from the application of a distance cutoff of 9 to
11\selectlanguage{ngerman}Å around the C\selectlanguage{greek}β \selectlanguage{english}atoms constitute the most accurate representation of
the visualized 3D structure. Nevertheless, the domain boundaries were
shown by a continuous domain curve (Figure 9B). This was followed by
heat map scores which showed a low domain shift point for AdeB protein
(Figure 9C). Overall, the predicted continuous domain protein secondary
structure of the AdeB suggested a confidence score of \textgreater{}8.6,
indicating our prediction was accurate.

\textbf{4 DISCUSSIONS}

A key part of the defense of \emph{A. baumannii} to different
antibiotics is a series of efflux pumps, which effectively exclude or
reduce the intracellular concentration of a large number of antibiotics
and other compounds, making the pathogen intrinsically more resistant to
some antimicrobial agents.\textsuperscript{30} The efflux pumps are a
topic of considerable interest, both from the perspective of
understanding functions and as targets for adjunct
therapies.\textsuperscript{45} In \emph{A. baumannii} , three RND efflux
systems, AdeABC, AdeFGH, and AdeIJK are reported to be primarily
associated with the emergence of MDR strains.\textsuperscript{16} Other
studies suggested that the AdeABC efflux pump system is the most
prevalent and with the highest detection rate in clinical isolates
of\emph{A. baumannii} as compared to other efflux pump
genes.\textsuperscript{11} Nevertheless, the RND efflux family
in\emph{A. baumannii} has broad substrate specificity and can efflux a
vast variety of antibiotics from the periplasm before the antibiotics
even fully enter the cell.\textsuperscript{46}

Despite a large number of identified efflux pumps, our understanding of
the molecular mechanisms drugs effluxes remain limited and relationships
between drugs and targets have been poorly
understood.\textsuperscript{47} The inhibitory mechanism between
amphiphilic peptide MP-B and the AdeB efflux pump remains
uncharacterized. Moreover, most of the current drug targets are
proteins, and the 3D structures of these proteins need to be known in
target identification.\textsuperscript{48} Therefore, Computer-aided
drug target identification methods can greatly reduce the searching
scope of experimental targets and associated costs by identifying the
drug binding sites and evaluating the druggability of the predicted
protein.\textsuperscript{47}

The results from this study provide novel insight into the inhibitory
activity of Mastoparan-B on the RND efflux pump. The preliminary
susceptibility test revealed that the organism was intrinsically
sensitive to MP-B as a potent inhibitor of the membrane function.
However, when the cells were grown in the presence of a sub-MIC
concentration of this peptide (0.5 \selectlanguage{greek}µ\selectlanguage{english}g/ml), and different concentrations
of antibiotics, there were significant reduction in MICs particularly
against aminoglycosides, fluoroquinolones, third-generation of
cephalosporins, carbapenems, and tetracycline, respectively. In
contrast, the MIC against colistin, piperacillin-tazobactam,
azithromycin, trimethoprim-sulfamethoxazole, and chloramphenicol
remained unchanged. This led us to the understanding that, the efflux
pump might be involved in this extrusion of particular antibiotics but
not all. The synergic activity of MP-B with antibiotics was further
supported by the 20 fold decrease in transcription of the \emph{ade} B
gene in the presence of sub-MIC of MP-B. Furthermore, the search for
phylogenetic analysis of AdeB protein with similar sequences in the
UniProtKB database revealed a high percent identity matrix using the
Maximum Likelihood method by neighbor joining method. The results
indicated the AdeB that investigated in this study was
evolutionary-related to the majority of Acinetobacter efflux pumps. This
was further confirmed by Structural neighbourhood comparison by CATH
database which showed 97.9\% alignment with functional family multidrug
efflux transporter AcrB transmembrane domain. The study of lineage of
the protein showed six functional families with the \emph{A. baumannii}
. Structural neighbourhood of superfamily (inside superfamily) indicated
evidence they have diverged from a common ancestor.

Furthermore, we performed docking of Mastoparan-B with amphiphilic
\selectlanguage{greek}α-\selectlanguage{english}helical structure to the inner membrane AdeB protein via the
amphiphobic amino acids. By AlphaFold 2 tool, we found that, the
proteins fold into a 3D dimensional shape to bury these hydrophobic side
chains in the protein interior. Since there was no reliable
crystallography for a membrane protein, so we relay on computational
methods. These methods are based on structural, physicochemical, and
evolutionary properties that distinguish binding sites from the rest of
the protein surface (e.g., amino acid composition and residue
conservation). Furthermore, we investigate deep learning of artificial
intelligence for the prediction of the membrane topology of
transmembrane proteins. The attachment of all forms of MP-B exclusively
occurred to the transmembrane helix-5 which is situated at the interior
of the inner membrane domain. This was confirmed by using the BioLiP
database (the structure was predicted by SPARKS-X (SPX) with template
7cz9A (z-score=33.51 and cutoff=6.9). Concurrent target-ligand
interaction indicated a shift in the dihedral angles with a lower free
binding energy score. Furthermore, examination of the dihedral angles
changes upon ligand binding showed that, the \selectlanguage{greek}φ \selectlanguage{english}and \selectlanguage{greek}ψ \selectlanguage{english}changes caused a
transient conformational change in the protein structure resulting in
the inhibition of AdeB activity. The presence of a large amount of Phe,
Ala, and Lys amino acid residues in the pore region provides a
hydrophobic trap around this peptide in the AdeB molecule particularly,
Phe residue was important in creating this hydrophobic microenvironment.
So far, the mechanisms have not been studied in detail, therefore this
statement must be further confirmed. Similarly, 3 inter domain
interchange was observed in AdeB \selectlanguage{greek}α-\selectlanguage{english}chains by FUpred software, utilizing
contact maps prediction created by deep residual neural networks coupled
with coevolutionary precision matrices.

\textbf{5 \textbar{} CONCLUSION}

AdeB is a critically important representative of the RND family of
multidrug transporters in \emph{A. baumannii} and as such, the molecular
interaction of this pump with Mastoparan-B by bioinformatics tools will
provide useful new information on the targeted inhibition of efflux
pumps in this bacterium. Mastoparan-B might be used in combination with
antibiotics like ampicillin-sulbactam for the treatment of MDR \emph{A.
baumannii} .

\textbf{AUTHORS' CONTRIBUTIONS}

S.MR performed the analysis and participated in the design of the study,
OZ, and OT, helped in writing and correcting of paper, FM helped drafted
the manuscript and analysis of data, and G.MM help with software
collection and information analysis. All authors read and approved the
final manuscript

\textbf{DATA AVAILABILITY STATEMENT}

The authors confirm that the data supporting the findings of this study
are available within the article and its supplementary materials.

\textbf{CONFLICTING INTERESTS}

The authors declare that they have no competing interests.

\textbf{FUNDING}

Not applicable.

\textbf{ORCID}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Mohammad Reza Shakibaie

https://orcid.org/0000-0003-2524-125X\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Farzan Modaresi

https://orcid.org/0000-0001-6253-7629\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Omid Azizi

https://orcid.org/0000-0001-7759-8029\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Omid Tadjrobehkar

https://orcid.org/0000-0002-5562-8070.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

Mohammad Mehdi Ghaemi

https://orcid.org/0000-0001-6427-2740

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\textbf{Figures legends}

\textbf{Figure. 1.} \textbf{A)} Antibiotic susceptibility of a
multidrug-resistant \emph{A. baumannii} isolates against 12 antibiotics
in the presence and absence of sub-MIC concentration (0.5 \selectlanguage{greek}µ\selectlanguage{english}g/ml) of
Mastoparan-B. The experiment was performed by broth microdilution test
as described by CLSI -2019. \textbf{B)} Growth curve analysis of
an\emph{A. baumannii} isolate in the presence and absence of sub-MIC of
MP-B. Values are presented as mean \selectlanguage{ngerman}± SD of three independent tests.

\textbf{Figure. 2. A)} Overview of the sequence/structure diversity of
the RND superfamily compared to other superfamilies in CATH (Class
Architecture Topology Homologous superfamily). The data indicated that
close superfamily relation among RND efflux pump using MDR efflux pump
ArcB. The red color circle indicate the position of our
family\textbf{B)} Unique species annotations and diversity by CATH
showed 91.8\% similarity among RND family, \textbf{C)} comparing the
lineage of superfamily of ArcB indicated at least 6 lineage identity
with \emph{A. baumannii} .

\textbf{Figure. 3.} \textbf{A)} Phylogenetic tree evaluation of AdeB
protein using UniProtKB database. For the phylogeny study we use
multiple amino acids alignment program and compared the sequences among
AdeB from different Acinetobacter sp. The percent identity was
calculated by determination of e-value from each sequence,
\textbf{B)}AdeB sequence parameters including Identity, Score, and
E-value of AdeB.\textbf{C)} Multiple amino acid alignment of helix-5 of
AdeB protein with closely related AdeB sequences in UniProtKB,
\textbf{D)} Percent identity matrix among closest sequences
(SD\textless{}99.4\%).

\textbf{Figure. 4.} Schematic of representative structures of RND
multidrug transporters and tripartite assemblies. \textbf{A)} Cartoon
structural prediction of the AdeABC RND efflux pump; a 30Å AdeC protein
containing \selectlanguage{greek}α/β \selectlanguage{english}barrel associated with a funnel and
H\textsuperscript{+}antiporter fusion (OMPA) activity extrude antibiotic
outside the cells. A 40\selectlanguage{romanian}Ǻ AdeA periplasmic protein with a heavy coil
structure in the form of coil-coil conformation, the AdeA was associated
with a large periplasmic cavity. A 50Ǻ the AdeB multidrug efflux pump
inner membrane protein. The AdeB inner transmembrane helix contained a
long narrow pore that connected to the central cavity. \textbf{B)} The
distal end of AdeB contains PC1, PN1, PC2, and PN2 subdomains with three
clefts.\textbf{C)} The wireframe diagram of the AdeABC RND efflux pump
system shows the AdeB as an inner transmembrane helix, a narrow pore
(rich in Phe, Lys, and Ala residues), a central cavity, a periplasmic
fusion protein (OMPA), and 3 chains ABC associated with RND component.
The docking site was shown by a circle.

\textbf{Figure. 5. A)} Ramachandran plot of AdeB protein based on
homology modeling of Expasy database. The MolProbity score and clash
points were estimated automatically. The majority of amino acids were in
favor region (Ɛ=0.79) which indicated they are in allowed regions for
backbone dihedral angles \selectlanguage{greek}ψ \selectlanguage{english}and \selectlanguage{greek}Φ \selectlanguage{english}of amino acid residues.
\textbf{B)}Local quality estimate of the chains A, B, and C of AdeB
protein using the Expasy program. \textbf{C)} ProSA Z-score of the AdeB
efflux pump protein. The score of +2.12 indicated correct conformation
of AdeB protein.

\textbf{Figure. 6. A)} The folding of transmembrane helix-5 and angstrom
changes in the amino acids involved in binding with MP-B (black arrow).
The correct folding at atomic level obtained by AlfaFold DB
program,\textbf{B)} A graphical representation of the shift in amino
acids angles (\selectlanguage{greek}ψ/\selectlanguage{english}F) by 9.0 \selectlanguage{ngerman}Å, 9.3 Å, and 9.6 Å. The arrow indicated
subatomic change in peptide bonds.

\textbf{Figure. 7} . \textbf{A)} Schematic illustration of the RND
tripartite efflux pump and AdeB binding site and internal domain chain
depicted by Expasy database and AlfaFold program, \textbf{B)} The
illustration of AdeB domain chain including transmembrane
helix-5,\textbf{C)} Amino acids in the helix-5 involved in molecular
docking with Mastoparan-B, \textbf{D)} Binding of the best pose of
\selectlanguage{greek}α-\selectlanguage{english}helix to MP-B (The ligand showed by a yellow color small circle),
\textbf{E)}Virtual position of amino acids form H-bounds attachment with
MP-B via using Autodock/Vina. The amino acids involved in docking are
shown in yellow color.

\textbf{Figure. 8. A)}Determination of the number and position of
transmembrane helices by the DeepTMHMM program
(http://dtu.biolib.com/DeepTMHMM/), \textbf{B)} Prediction of AdeB
topology by the TOPOCONS software
(https://topcons.cbr.su.se/),\textbf{C)} Percent of, inside,
transmembrane, and outside helices using the TOPOCONS tool.

\textbf{Figure. 9.} \textbf{A)} Contact map measured by FUpred software
for accuracy of AdeB domains, \textbf{B)} Fu score for domains
boundaries and \textbf{C)} FU score for heat map which shows domain
shifting point of the AdeB efflux pump protein\textbf{.} The axes marked
the residue index along the sequence in the contact map. For the contact
map, each dot represents a residue pair with predicted contact. The
terminal contacts and the corresponding inter-chain interaction are
highlighted with a black box.\selectlanguage{english}
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\textbf{Hosted file}

\verb`Table.docx`  available at \url{https://authorea.com/users/567780/articles/613951-conformational-changes-in-the-adeb-transmembrane-efflux-pump-by-amphiphilic-peptide-mastoparan-b-down-regulates-expression-of-the-ade-b-gene-and-restores-antibiotics-susceptibility}

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