1 INTRODUCTION
Computation chemistry has greatly increased our knowledge about protein conformation and drug interaction and helped us better understand microbial infection treatment.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.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.4
In this regard, 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.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.6 A. baumannii has different resistance mechanisms against antimicrobial agents such as β-Lactam inactivation, biofilm formation, efflux pumps, alterations of the affinity for antibiotics, etc.7-9One 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.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).12 The best-characterized multi-drug efflux system in A. baumannii is the RND superfamily.13 These tripartite complexes comprise an inner membrane that acts as a secondary active H+/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).14 The primary RND efflux pump of A. baumannii (AdeABC) was first described in MDR strain BM4454.14 The 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.15The AdeB is an inner membrane protein and transports various drugs through the central pore.16 The pore is opened to the periplasm via three vestibules located at subunit interfaces and a central cavity.17 More researchers demonstrated that, the ade B is the most important gene in the adeABC efflux system.17,18 Genetic studies suggested that discrimination between the substrates in A. baumannii occurs mainly within the periplasmic region instead of the transmembrane region.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.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.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.22 Membrane proteins are difficult to study due to their partially hydrophobic surfaces, flexibility, and lack of stability.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.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.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.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.26 These compounds frequently destabilize biological membranes by dual amphiphilic activity resulting in membrane perforation.27 In recent years, numerous efflux pump inhibitors have been discovered and tested, including natural products, antibiotics, and synthetic molecules.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 Escherichia coli AcrAB-TolC and thePseudomonas aeruginosa MexAB-OprM complexes were well delineated.29 In contrast, there is no information about the effect of Mastoparan-B on the RND efflux in A. baumannii . It has been reported that the phenylalanine-arginine β-naphthylamide (PAβN) a broad-spectrum efflux pump inhibitor interacts with AdeB strongly through the distal binding pocket.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 ofA. baumannii .31
Therefore, in the present study, for the first time, we are reporting transcriptional analysis of the 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.