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

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB
Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB
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.
Reading, Eamonn
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Ahdash, Zainab
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Fais, Chiara
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Ricci, Vito
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Kan, Xuan Wang
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Grimsey, Elizabeth
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Stone, Jack
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Malloci, Giuliano
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Lau, Andy M.
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Findlay, Heather
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Konijnenberg, Albert
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Booth, Paula J.
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Ruggerone, Paolo
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Vargiu, Attilio V.
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Piddock, Laura J. V.
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Politis, Argyris
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Reading, Eamonn
62fed933-f867-4c72-89e7-83aea573a836
Ahdash, Zainab
13e241fc-e13f-444c-842f-87da95d7143b
Fais, Chiara
87a0cc5d-e124-404a-8df2-3ab8b9c250e7
Ricci, Vito
8df7818c-6a71-4628-879d-d647a3650571
Kan, Xuan Wang
f4aed380-ba35-4707-b0b4-8bc36ba31a1a
Grimsey, Elizabeth
d4c76de4-4543-430a-8c77-1b2e9cac5945
Stone, Jack
b91138b7-4394-4b11-b63b-9a29c0ede487
Malloci, Giuliano
4b3d7a51-95c6-4178-821f-ee83fc9c43bd
Lau, Andy M.
56ef41c9-3268-40e2-b693-b54a334f358c
Findlay, Heather
110898ff-7acb-4642-a96d-c6e031488efd
Konijnenberg, Albert
9fe77173-0147-4933-bf02-6431b6f27e87
Booth, Paula J.
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Ruggerone, Paolo
d4f67f89-3252-465a-bc9c-9c81c0eeb6f4
Vargiu, Attilio V.
efa6a2ab-5666-422a-9e3a-60cfc853908c
Piddock, Laura J. V.
cefb6727-1a1a-4109-8424-e33fa88d3ff9
Politis, Argyris
06febde7-4b5c-4435-9b3a-8915f50c47a3

[Unknown type: UNSPECIFIED]

Record type: UNSPECIFIED

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.

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Published date: 28 April 2020

Identifiers

Local EPrints ID: 479166
URI: http://eprints.soton.ac.uk/id/eprint/479166
PURE UUID: 995b404b-5d74-4a40-9206-1f082c0bfb1e
ORCID for Eamonn Reading: ORCID iD orcid.org/0000-0001-8219-0052

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Date deposited: 20 Jul 2023 16:40
Last modified: 17 Mar 2024 04:19

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Contributors

Author: Eamonn Reading ORCID iD
Author: Zainab Ahdash
Author: Chiara Fais
Author: Vito Ricci
Author: Xuan Wang Kan
Author: Elizabeth Grimsey
Author: Jack Stone
Author: Giuliano Malloci
Author: Andy M. Lau
Author: Heather Findlay
Author: Albert Konijnenberg
Author: Paula J. Booth
Author: Paolo Ruggerone
Author: Attilio V. Vargiu
Author: Laura J. V. Piddock
Author: Argyris Politis

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