title: Direct interaction of multidrug efflux transporter AcrB and outer membrane channel TolC detected via site-directed disulfide cross-linking.
abstract: The AcrAB-TolC system exports a wide variety of drugs and toxic compounds, and confers intrinsic drug tolerance on Escherichia coli. The crystal structures suggested that AcrB and TolC directly dock with each other. However, biochemical and biophysical evidence of their interaction has been contradictory until recently. In this study, we examine the interaction sites by means of in vivo disulfide cross-linking between cysteine residues introduced by site-directed mutagenesis at the tops of the vertical hairpins of AcrB and the bottoms of the coiled coils of polyhistidine-tagged TolC molecules, which are structurally predicted docking sites. The AcrB-TolC complex formed through disulfide cross-linking was detected when a specific pair of mutants was coexpressed in E. coli. Our observations suggested that the AcrB-TolC complex may be formed through a two-step mechanism via transient tip-to-tip interaction of AcrB and TolC. The cross-linking was not affected by AcrA, the substrate, or a putative proton coupling site mutation.
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Norihisa
authors.initials: N
authors.lastname: Tamura
authors.forename: Satoshi
authors.initials: S
authors.lastname: Murakami
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Oyama
authors.forename: Masaji
authors.initials: M
authors.lastname: Ishiguro
authors.forename: Akihito
authors.initials: A
authors.lastname: Yamaguchi
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004361:Drug Tolerance / Q000502:physiology; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D020836:Protein Structure, Quaternary / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003545:Cysteine; D004220:Disulfides; D029968:Escherichia coli Proteins; D006639:Histidine; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D046912:Multiprotein Complexes; C083496:AcrB protein, E coli; C073126:tolC protein, E coli; C033223:polyhistidine