title: pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efflux system.
abstract: The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.
authors.affiliation: Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Hermia
authors.initials: H
authors.lastname: Ip
authors.forename: Kelly
authors.initials: K
authors.lastname: Stratton
authors.forename: Helen
authors.initials: H
authors.lastname: Zgurskaya
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
mesh_terms: D000409:Alanine / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D024901:Drug Resistance, Multiple, Bacterial; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008074:Lipoproteins / Q000737:chemistry*; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins; D008956:Models, Chemical; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D013053:Spectrophotometry; D013696:Temperature
chemical_list: D000409:Alanine; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003545:Cysteine; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C073126:tolC protein, E coli