Figures legends
Figure. 1. A) Antibiotic susceptibility of a
multidrug-resistant A. baumannii isolates against 12 antibiotics
in the presence and absence of sub-MIC concentration (0.5 µg/ml) of
Mastoparan-B. The experiment was performed by broth microdilution test
as described by CLSI -2019. B) Growth curve analysis of anA. baumannii isolate in the presence and absence of sub-MIC of
MP-B. Values are presented as mean ± SD of three independent tests.
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 familyB) Unique species annotations and diversity by CATH showed
91.8% similarity among RND family, C) comparing the lineage of
superfamily of ArcB indicated at least 6 lineage identity with A.
baumannii .
Figure. 3. 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, B)AdeB sequence parameters including Identity, Score, and E-value of AdeB.C) Multiple amino acid alignment of helix-5 of AdeB protein
with closely related AdeB sequences in UniProtKB, D) Percent
identity matrix among closest sequences (SD<99.4%).
Figure. 4. Schematic of representative structures of RND
multidrug transporters and tripartite assemblies. A) Cartoon
structural prediction of the AdeABC RND efflux pump; a 30Å AdeC protein
containing α/β barrel associated with a funnel and H+antiporter fusion (OMPA) activity extrude antibiotic outside the cells.
A 40Ǻ 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. B) The distal end of AdeB
contains PC1, PN1, PC2, and PN2 subdomains with three clefts.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.
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 ψ and Φ of amino acid residues. B)Local quality estimate of the chains A, B, and C of AdeB protein using
the Expasy program. C) ProSA Z-score of the AdeB efflux pump
protein. The score of +2.12 indicated correct conformation of AdeB
protein.
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,B) A graphical representation of the shift in amino acids
angles (ψ/ɸ) by 9.0 Å, 9.3 Å, and 9.6 Å.
The arrow indicated subatomic
change in peptide bonds.
Figure. 7 . A) Schematic illustration of the RND
tripartite efflux pump and AdeB binding site and internal domain chain
depicted by Expasy database and AlfaFold program, B) The
illustration of AdeB domain chain including transmembrane helix-5,C) Amino acids in the helix-5 involved in molecular docking
with Mastoparan-B, D) Binding of the best pose of α-helix to
MP-B (The ligand showed by a yellow color small circle), 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.
Figure. 8. A)Determination of the number and
position of transmembrane helices by the DeepTMHMM program
(http://dtu.biolib.com/DeepTMHMM/), B) Prediction of AdeB
topology by the TOPOCONS software (https://topcons.cbr.su.se/),C) Percent of, inside, transmembrane, and outside helices using
the TOPOCONS tool.
Figure. 9. A) Contact map measured by FUpred software
for accuracy of AdeB domains, B) Fu score for domains
boundaries and C) FU score for heat map which shows domain
shifting point of the AdeB efflux pump protein. 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.