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

Abstract

Resistance-nodulation-division (RND) efflux pumps play a key role in inherent and evolved multidrug-resistance (MDR) in bacteria. AcrB is the prototypical member of the RND family and acts to recognise and export a wide range of chemically distinct molecules out of bacteria, conferring resistance to a variety of antibiotics. Although high resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown, preventing a complete mechanistic understanding of efflux and inhibition. Here, we determine these structural dynamics in the presence of AcrB substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular modelling, drug binding and bacterial susceptibility studies. We show that the well-studied efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) 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 an MDR clinical isolate, AcrBG288D, modifies the plasticity of the transport pathway, which could explain its altered substrate specificity. Our results provide molecular insight into drug export and inhibition of a major MDR-conferring efflux pump and the important directive role of its dynamics.

Cryo-EM Structure and Molecular Dynamics Analysis of the Fluoroquinolone Resistant Mutant of the AcrB Transporter from Salmonella

“…Thus, the G288D mutation appears to impact the plasticity of the drug-binding pocket and drug-transport pathway. These results are further corroborated by the recent analysis of S TmAcrB G288D sensitivity to another EPI, namely phenylalanine-arginine-β-naphthylamide (PAβN) [ 79 ], which not only demonstrates that PAβN effectively inhibits both WT and G288D version of the Salmonella AcrB, but that that ciprofloxacin and PAβN can stably occupy the DBP at the same time. This implies that PAβN potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding.…”