Tripartite efflux pumps of the RND superfamily: what did we learn from computational studies?

Abstract

Bacterial resistance to antibiotics has been long recognized as a priority to address for human health. Among all micro-organisms, the so-called multi-drug resistant (MDR) bacteria, which are resistant to most, if not all drugs in our current arsenal, are particularly worrisome. The World Health Organization has prioritized the ESKAPE ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa and Enterobacter species) pathogens, which include four Gram-negative bacterial species. In these bacteria, active extrusion of antimicrobial compounds out of the cell by means of ‘molecular guns’ known as efflux pumps is a main determinant of MDR phenotypes. The resistance-nodulation-cell division (RND) superfamily of efflux pumps connecting the inner and outer membrane in Gram-negative bacteria is crucial to the onset of MDR and virulence, as well as biofilm formation. Thus, understanding the molecular basis of the interaction of antibiotics and inhibitors with these pumps is key to the design of more effective therapeutics. With the aim to contribute to this challenge, and complement and inspire experimental research, in silico studies on RND efflux pumps have flourished in recent decades. Here, we review a selection of such investigations addressing the main determinants behind the polyspecificity of these pumps, the mechanisms of substrate recognition, transport and inhibition, as well as the relevance of their assembly for proper functioning, and the role of protein–lipid interactions. The journey will end with a perspective on the role of computer simulations in addressing the challenges posed by these beautifully complex machineries and in supporting the fight against the spread of MDR bacteria.

Structure of the Bacterial Cell Envelope and Interactions with Antimicrobials: Insights from Molecular Dynamics Simulations

“…Recent additions to the literature on peptidoglycan model development for both Gram-positive and Gram-negative strains as well as attempts to model the complex landscape of the periplasm have been addressed. We have not discussed literature concerning the transmembrane protein machinery involved in lipid transport and folding as well as efflux pump action, and recent reviews , cover the literature in this field.…”

Titratable residues that drive RND efflux: Insights from molecular simulations

“…Computational studies have been widely used to investigate efflux machinery (Athar et al, 2023 ). Early simulations did not focus explicitly on the proton relay network, instead utilising postulated protonation states (Seeger et al, 2008 ).…”

“…Further simulations by the same group displayed flexibility in the porter domain, with opening and closing of the periplasmic cleft (Fischer and Kandt, 2013 ). For an extensive discussion of RND simulation studies, we direct the reader to a recent review (Athar et al, 2023 ). Here we turn our attention only to simulation studies where the protonation states of the proton relay network residues have been a primary focus.…”

Vibrio cholerae RND efflux systems: mediators of stress responses, colonization and pathogenesis

“…Crystallized protein domains of RND efflux systems in the presence and absence of substrates or inhibitors provided a wealth of information on assembly and function [see ( Alav et al., 2021 ; Athar et al., 2023 )]. Analyses of the crystal structures of multiple RND transporter homologues revealed that RND pump proteins are structurally conserved; they all include multiple substrate entry portals and several substrate binding sites which mediate uptake of substrates from the periplasm and from within the cytoplasmic membrane.…”