title: Functional relevance of AcrB Trimerization in pump assembly and substrate binding.
abstract: AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhong
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Qian
authors.initials: Q
authors.lastname: Chai
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Zhaoshuai
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Linliang
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D055786:Gene Knockout Techniques; D026901:Membrane Transport Proteins / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D003545:Cysteine; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; C083496:AcrB protein, E coli; C073126:tolC protein, E coli