PMID:16101295
| Citation |
Tamura, N, Murakami, S, Oyama, Y, Ishiguro, M and Yamaguchi, A (2005) Direct interaction of multidrug efflux transporter AcrB and outer membrane channel TolC detected via site-directed disulfide cross-linking. Biochemistry 44:11115-21 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi050452u |
| Keywords |
Amino Acid Substitution/genetics; Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Cell Membrane/genetics; Cell Membrane/metabolism; Cysteine/chemistry; Cysteine/genetics; Cysteine/metabolism; Disulfides/chemistry; Disulfides/metabolism; Drug Tolerance/physiology; Escherichia coli/chemistry; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Expression; Histidine/chemistry; Histidine/genetics; Histidine/metabolism; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/metabolism; Membrane Transport Proteins; Multidrug Resistance-Associated Proteins; Multiprotein Complexes/chemistry; Multiprotein Complexes/genetics; Multiprotein Complexes/metabolism; Point Mutation; Protein Binding/genetics; Protein Structure, Quaternary/genetics; Protein Structure, Secondary/genetics |
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