Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

Benjamin Russell Lewis; Muhammad R. Uddin; Mohammad Moniruzzaman; Katie M. Kuo; Anna J. Higgins; Laila M.N. Shah; Frank Sobott; Jerry M. Parks; Dietmar Hammerschmid; James C. Gumbart; Helen I. Zgurskaya; Eamonn Reading

doi:10.1038/s41467-023-39615-x

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

Benjamin Russell Lewis, Muhammad R. Uddin, Mohammad Moniruzzaman, Katie M. Kuo, Anna J. Higgins, Laila M.N. Shah, Frank Sobott, Jerry M. Parks, Dietmar Hammerschmid, James C. Gumbart, Helen I. Zgurskaya, Eamonn Reading

Molecular Biophysics

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

Original language	English
Article number	3900
Journal	Nature Communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-39615-x
State	Published - Dec 2023

Funding

Work at King’s College London was supported by a UKRI Future Leaders Fellowship (MR/S015426/1) to E.R. and a King’s College London PhD studentship to B.R.L. This work was also supported by US National Institutes of Health grant R01-AI052293 to H.I.Z., J.M.P., and J.C.G. Computational resources were provided through XSEDE (TG-MCB130173), which is supported by the US National Science Foundation (NSF; ACI-1548562). This research used resources at the Compute and Data Environment for Science (CADES) at ORNL, which is managed by UT Battelle, LLC, for DOE under contract DE-AC05–00OR22725. This work also used the Hive cluster, which is supported by the NSF (1828187) and is managed by the Partnership for an Advanced Computing Environment (PACE) at GT. The Q-Exactive Plus UHMR at the University of Leeds used for native MS was funded by The Wellcome Trust (208385/Z/17/Z). A.J.H. was funded by a BBSRC IPA grant (BB/R018561/1/) in collaboration with GSK and UCB Pharma. The authors thank Valeria Calvaresi for advice with HDX analysis and statistics.

Funders	Funder number
BBSRC IPA	BB/R018561/1/
CADES
Data Environment for Science
XSEDE	TG-MCB130173
National Science Foundation
National Institutes of Health	R01-AI052293
U.S. Department of Energy	1828187, DE-AC05–00OR22725
King’s College London
Wellcome Trust	208385/Z/17/Z
UK Research and Innovation	MR/S015426/1
Neurosciences Foundation	ACI-1548562

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title = "Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein",

abstract = "Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.",

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AU - Shah, Laila M.N.

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AU - Parks, Jerry M.

AU - Hammerschmid, Dietmar

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AU - Zgurskaya, Helen I.

AU - Reading, Eamonn

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AB - Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

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Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

Abstract

Funding

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