Binding and Transport of Carboxylated Drugs by the Multidrug Transporter AcrB

AcrAB(Z)-TolC is the main drug efflux transporter complex in Escherichia coli. The extrusion of various toxic compounds depends on several drug binding sites within the trimeric AcrB transporter. Membrane-localized carboxylated substrates, such as fusidic acid and hydrophobic β-lactams, access the p...

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Bibliographic Details
Published in:	Journal of molecular biology Vol. 432; no. 4; pp. 861 - 877
Main Authors:	Tam, Heng-Keat, Malviya, Viveka N., Foong, Wuen-Ee, Herrmann, Andrea, Malloci, Giuliano, Ruggerone, Paolo, Vargiu, Attilio V., Pos, Klaas M.
Format:	Journal Article
Language:	English
Published:	England Elsevier Ltd 14-02-2020
Subjects:	Binding Sites Chromatography, Gel crystallography drug transport efflux Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - chemistry Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism membrane protein Microbial Sensitivity Tests Models, Theoretical Molecular Dynamics Simulation molecular modeling Multidrug Resistance-Associated Proteins - chemistry Multidrug Resistance-Associated Proteins - genetics Multidrug Resistance-Associated Proteins - metabolism Protein Conformation drug transport membrane protein crystallography efflux molecular modeling
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Summary:	AcrAB(Z)-TolC is the main drug efflux transporter complex in Escherichia coli. The extrusion of various toxic compounds depends on several drug binding sites within the trimeric AcrB transporter. Membrane-localized carboxylated substrates, such as fusidic acid and hydrophobic β-lactams, access the pump via a groove between the transmembrane helices TM1 and TM2. In this article, the transport route from the initial TM1/TM2 groove binding site toward the deep binding pocket located in the periplasmic part has been addressed via molecular modeling studies followed by functional and structural characterization of several AcrB variants. We propose that membrane-embedded drugs bind initially to the TM1/TM2 groove, are oriented by the AcrB PN2 subdomain, and are subsequently transported via a PN2/PC1 interface pathway directly toward the deep binding pocket. Our work emphasizes the exploitation of multiple transport pathways by AcrB tuned to substrate physicochemical properties related to the polyspecificity of the pump. [Display omitted]
Bibliography:	Author contributions: Heng-Keat Tam: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – Original Draft, Writing – Review & Editing, Visualization. Viveka N. Malviya: Methodology, Validation, Formal analysis, Investigation, Data curation, Visualization. Wuen-Ee Foong: Methodology, Validation, Formal analysis, Investigation, Data curation, Visualization. Andrea Herrmann: Investigation. Giuliano Malloci: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – Original Draft, Writing – Review & Editing. Paolo Ruggerone: Conceptualization, Methodology, Validation, Resources, Writing – Review & Editing, Supervision, Funding acquisition. Attilio V. Vargiu: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – Original Draft, Writing – Review & Editing, Supervision. Klaas M. Pos: Conceptualization, Methodology, Validation, Formal analysis, Resources, Data curation, Writing – Original Draft, Writing – Review & Editing, Supervision, Project administration, Funding acquisition. Author contributions: H.K.T., P.R., G.M., A.V.V., and K.M.P. designed research. H.K.T. performed the protein purification, crystallization, and diffraction data collection. H.K.T. and W.E.F. performed the in vivo functional experiments. A.H. performed protein production and protein purification. V.N.M. performed the ITC experiments. H.K.T. and K.M.P. determined the AcrB variant crystal structures. G.M., A.V.V. and P.R. performed computational work. H.K.T., V.N.M., W.E.F., G.M., A.V.V., P.R. and K.M.P. wrote the manuscript. All authors contributed to data analysis.
ISSN:	0022-2836 1089-8638
DOI:	10.1016/j.jmb.2019.12.025