PMID:19411330
| Citation |
Ge, Q, Yamada, Y and Zgurskaya, H (2009) The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC. J. Bacteriol. 191:4365-71 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC2698478 Online version:10.1128/JB.00204-09 |
| Keywords |
Anti-Bacterial Agents/pharmacology; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli/drug effects; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Immunoblotting; Lipoproteins/chemistry; Lipoproteins/genetics; Lipoproteins/metabolism; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Microbial Sensitivity Tests; Multidrug Resistance-Associated Proteins/genetics; Multidrug Resistance-Associated Proteins/metabolism; Mutagenesis, Site-Directed; Protein Structure, Tertiary/genetics; Protein Structure, Tertiary/physiology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship; Trypsin/metabolism |
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