Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump.
AcrAB-TolC is the major, constitutively expressed efflux protein complex that provides resistance to a variety of antimicrobial agents in Escherichia coli. Previous studies showed that AcrA, a periplasmic protein of the membrane fusion protein family, could function with at least two other resistance-nodulation-division family pumps, AcrD and AcrF, in addition to its cognate partner, AcrB. We found that, among other E. coli resistance-nodulation-division pumps, YhiV, but not MdtB or MdtC, could also function with AcrA. When AcrB was assessed for the capacity to function with AcrA homologs, only AcrE, but not YhiU or MdtA, could complement an AcrA deficiency. Since AcrA could, but YhiU could not, function with AcrB, we engineered a series of chimeric mutants of these proteins in order to determine the domain(s) of AcrA that is required for its support of AcrB function. The 290-residue N-terminal segment of the 398-residue protein AcrA could be replaced with a sequence coding for the corresponding region of YhiU, but replacement of the region between residues 290 and 357 produced a protein incapable of functioning with AcrB. In contrast, the replacement of residues 357 through 397 of AcrA still produced a functional protein. We conclude that a small region of AcrA close to, but not at, its C terminus is involved in the interaction with its cognate pump protein, AcrB.
Elkins
Christopher A
CA
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.
Nikaido
Hiroshi
H
D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000502:physiology; D029968:Escherichia coli Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D008074:Lipoproteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C434281:Mdt-A protein, E coli; C477834:MdtE protein, E coli; C477833:MdtF protein, E coli