Gram-negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance-nodulation-cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB-TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/ AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single-site substitutions and highlight regions that are more sensitive to perturbation.

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

Multidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For the resistance nodulation and cell division (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system from Escherichia coli, drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å 2 are less well transported than other substrates. The Escherichia coli AcrAB-TolC tripartite multidrug efflux system confers resistance against a wide variety of drug compounds, including antibiotics, dyes, organic solvents, and detergents (1-3). The pump utilizes the proton motive force in an antiport fashion to energize the extrusion of compounds from the periplasm of the Gram-negative cell to the extracellular environment. Drug transport and energy transduction are catalyzed by the homotrimeric inner membrane component AcrB, belonging to the large superfamily of resistance nodulation and cell division (RND) transporters. AcrB contains two large periplasmic loops between transmembrane helices 1 and 2 plus 7 and 8. The protomers within the asymmetric trimeric setup adopt three different states, designated loose (L), tight (T), and open (O) (Fig. 1) (4-6). Recent crystallographic analysis of AcrB in complex with antibiotic molecules indicated that large drug molecules bind to the periplasmic access pocket (AP) of the protomer in the L state (7,8). These and earlier studies (4) provided further insight into drug binding into the periplasmic deep binding pocket (DBP) in the protomer adopting the T state. Since only a few structures of AcrB in complex with antibiotics (minocyclin, doxorubicin, rifampin, erythromycin, and a pyridopyrimidine derivative inhibitor [4,[7][8][9]) have yet been obtained, it remains elusive whether the AP and DBP can bind the same drugs. Nakashima et al. (8) showed an AcrB trimer structure with rifampin bound in the L monomer in addition to binding of minocycline in the T monomer, and Eicher et al. (7) showed a trimer structure with doxorubicin bound to both the DBP and AP, with the latter occupied by a doxorubicin dimer. The question therefore arises whether these pockets represent sequential binding sites within the rotational transport cycle and if both pockets bind the same set of drugs. Both studies (7, 8) also indicated that a flexible switch loop of 11 amino acid residues residing between the AP and DBP might play an essential role in drug transport during the conformational cycling of the protomers through the L, T, and O states. Comparison ...

Antimicrobial resistance (AMR) has become a major problem in public health leading to an estimated 4.95 million deaths in 2019. The selective pressure caused by the massive and repeated use of antibiotics has led to bacterial strains that are partially or even entirely resistant to known antibiotics. AMR is caused by several mechanisms, among which the (over)expression of multidrug efflux pumps plays a central role. Multidrug efflux pumps are transmembrane transporters, naturally expressed by Gram-negative bacteria, able to extrude and confer resistance to several classes of antibiotics. Targeting them would be an effective way to revive various options for treatment. Many efflux pump inhibitors (EPIs) have been described in the literature; however, none of them have entered clinical trials to date. This review presents eight families of EPIs active against Escherichia coli or Pseudomonas aeruginosa. Structure–activity relationships, chemical synthesis, in vitro and in vivo activities, and pharmacological properties are reported. Their binding sites and their mechanisms of action are also analyzed comparatively.

The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.

In Gram-negative bacteria, tripartite efflux pumps, like AcrAB-TolC from Escherichia coli, play a prominent role in the resistance against multiple antibiotics. Transport of the drugs across the outer membrane and its coupling to the electrochemical gradient is dependent on the presence of all three components. As the activity of the E. coli AcrAB-TolC efflux pump is dependent on both the concentration of substrates and the extent of the electrochemical gradient across the inner membrane, the dynamics of tripartite pump assembly and disassembly might be crucial for effective net transport of drugs towards the outside of the cell.