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.