The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC.
Periplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain of Escherichia coli AcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump.
Ge
Qiang
Q
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Yamada
Yoichi
Y
Zgurskaya
Helen
H
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008074:Lipoproteins / 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 / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014357:Trypsin; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C073126:tolC protein, E coli