TY - JOUR
T1 - Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein
AU - Russell Lewis, Ben
AU - Uddin , Muhammad R.
AU - Moniruzzaman, Mohammad
AU - Kuo, Katie M.
AU - Higgins, Anna J.
AU - Shah, Laila
AU - Sobott, Frank
AU - Parks, Jerry
AU - Hammerschmid, Dietmar
AU - Gumbart, James
AU - Zgurskaya , Helen I.
AU - Reading, Eamonn
N1 - 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.
Publisher Copyright:
© 2023, The Author(s).
PY - 2023/7/18
Y1 - 2023/7/18
N2 - 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.
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.
KW - Antimicrobial resistance
KW - efflux pump inhibitor
KW - hydrogen deuterium exchange (HDX) mass spectrometry
KW - Membrane proteins
UR - http://www.scopus.com/inward/record.url?scp=85165281575&partnerID=8YFLogxK
U2 - 10.1038/s41467-023-39615-x
DO - 10.1038/s41467-023-39615-x
M3 - Article
SN - 2041-1723
VL - 14
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 3900
ER -