title: Genetic assessment of the role of AcrB β-hairpins in the assembly of the TolC-AcrAB multidrug efflux pump of Escherichia coli.
abstract: The tripartite AcrAB-TolC multidrug efflux pump of Escherichia coli is the central conduit for cell-toxic compounds and contributes to antibiotic resistance. While high-resolution structures of all three proteins have been solved, much remains to be learned as to how the individual components come together to form a functional complex. In this study, we investigated the importance of the AcrB β-hairpins belonging to the DN and DC subdomains, which are presumed to dock with TolC, in complex stability and activity of the complete pump. Our data show that the DN subdomain β-hairpin residues play a more critical role in complex stability and activity than the DC subdomain hairpin residues. The failure of the AcrB DN β-hairpin deletion mutant to engage with TolC leads to the drug hypersensitivity phenotype, which is reversed by compensatory alterations in the lipoyl and β-barrel domains of AcrA. Moreover, AcrA and TolC mutants that induce TolC opening also reverse the drug hypersensitivity phenotype of the AcrB β-hairpin mutants, indicating a failure by the AcrB mutant to interact and thus induce TolC opening on its own. Together, these data suggest that both AcrB β-hairpins and AcrA act to stabilize the tripartite complex and induce TolC opening for drug expulsion.
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, AZ, 85287, USA; Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, OK, 73019, USA.
authors.forename: Jon W
authors.initials: JW
authors.lastname: Weeks
authors.forename: Vassiliy N
authors.initials: VN
authors.lastname: Bavro
authors.forename: Rajeev
authors.initials: R
authors.lastname: Misra
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic
chemical_list: D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; 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