Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein
Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein
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.
Anti-Bacterial Agents/pharmacology, Bacterial Outer Membrane Proteins/metabolism, Biological Transport, Escherichia coli Proteins/metabolism, Escherichia coli/metabolism, Membrane Transport Proteins/metabolism, Multidrug Resistance-Associated Proteins/metabolism
Russell Lewis, Benjamin
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Uddin, Muhammad R.
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Moniruzzaman, Mohammad
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Kuo, Katie M.
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Higgins, Anna J.
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Shah, Laila M.N.
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Sobott, Frank
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Parks, Jerry M.
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Hammerschmid, Dietmar
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Gumbart, James C.
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Zgurskaya, Helen I.
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Reading, Eamonn
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18 July 2023
Russell Lewis, Benjamin
6052ce96-6af8-4c50-a49c-594782e76493
Uddin, Muhammad R.
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Moniruzzaman, Mohammad
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Kuo, Katie M.
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Higgins, Anna J.
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Shah, Laila M.N.
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Sobott, Frank
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Parks, Jerry M.
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Hammerschmid, Dietmar
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Gumbart, James C.
6cdffebe-215c-411a-a4ba-51d7796c9f93
Zgurskaya, Helen I.
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Reading, Eamonn
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Russell Lewis, Benjamin, Uddin, Muhammad R., Moniruzzaman, Mohammad, Kuo, Katie M., Higgins, Anna J., Shah, Laila M.N., Sobott, Frank, Parks, Jerry M., Hammerschmid, Dietmar, Gumbart, James C., Zgurskaya, Helen I. and Reading, Eamonn
(2023)
Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein.
Nature Communications, 14, [3900].
(doi:10.1038/s41467-023-39615-x).
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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Conformational restriction shapes the inhibition of a multidrug
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Accepted/In Press date: 15 June 2023
Published date: 18 July 2023
Additional Information:
Funding Information:
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.
Keywords:
Anti-Bacterial Agents/pharmacology, Bacterial Outer Membrane Proteins/metabolism, Biological Transport, Escherichia coli Proteins/metabolism, Escherichia coli/metabolism, Membrane Transport Proteins/metabolism, Multidrug Resistance-Associated Proteins/metabolism
Identifiers
Local EPrints ID: 480809
URI: http://eprints.soton.ac.uk/id/eprint/480809
ISSN: 2041-1723
PURE UUID: b727e390-7eca-4328-97ce-224ab39dadde
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Date deposited: 09 Aug 2023 17:16
Last modified: 17 Mar 2024 04:19
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Contributors
Author:
Benjamin Russell Lewis
Author:
Muhammad R. Uddin
Author:
Mohammad Moniruzzaman
Author:
Katie M. Kuo
Author:
Anna J. Higgins
Author:
Laila M.N. Shah
Author:
Frank Sobott
Author:
Jerry M. Parks
Author:
Dietmar Hammerschmid
Author:
James C. Gumbart
Author:
Helen I. Zgurskaya
Author:
Eamonn Reading
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