Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB

Abstract

Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.

Pyridylpiperazine-based allosteric inhibitors of RND-type multidrug efflux pumps

“…Each system was first subjected to a multi-step structural relaxation via a combination of steepest descent (5000 steps) and conjugate gradient (up to 25000 steps) methods using the pmemd program implemented in AMBER18, as described previously 29,33,38,59,60,[66][67][68] . The systems were then heated from 0 to 310 K in two steps: (i) from 0 to 100 K in 1 ns under constant-volume conditions, with harmonic restraints (k = 1 kcal•mol −1 •Å −2 ) applied to the heavy atoms of both the protein and the lipids; (ii) from 100 to 310 K in 5 ns under constant pressure (set to a value of 1 atm) with restraints on the heavy atoms of the protein and on the z coordinates of the phosphorous atoms of the lipids, so as to allow membrane rearrangement during heating.…”

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“…Computational and experimental data suggested that ciprofloxacin and PAβN bind to AcrB simultaneously at different subsites within the distal binding pocket and that PAβN inhibits efflux by perturbing the substrate translocation pathway. 885 PAβN was tested in vivo to be cytotoxic. 886 However, it is still one of the most commonly used inhibitors in assays to restore the activity of various antibiotics.…”

Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus

“…Unlike higher resolution approaches, HDX-MS offers the advantage of obtaining dynamic structural information for samples that presents heterogeneous and/or flexible areas. It was successfully applied to characterize the dynamics and interactions of membrane transporters and even GPCRs (48)(49)(50)(51)(52)(53).…”