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Insight into the Multidrug‐Efflux Pump AcrB Oligomerization Process

AcrA‐AcrB‐TolC is a three component efflux pump of Escherichia coli. It allows the extrusion of a wide range of cytotoxic compounds. AcrB is located in the inner membrane of the bacterium. It is a homotrimer and utilizes the proton gradient across the inner membrane to drive the export of drugs. In this study, we tested the hypothesis that the intertwining loop of AcrB (residues 211–240) is critical for the assembly of AcrB trimer. Expression and activity assay of AcrB were conducted in an E. coli strain deficient in acrB (ΔacrB). Site‐directed mutagensis studies of the loop region revealed that several residues in the loop were critical for AcrB function. To compare the conformation of the loop in the wild type and mutant AcrB, a cysteine‐pair was introduced in the middle of the loop. The extent of disulfide bond formation was used as a reporter to probe structural changes in the function‐less mutants. Finally, the oligomeric state of the purified mutated protein was determined using blue native polyacrylamide gel electrophoresis (BN‐PAGE). Our results suggest that the loop of AcrB assumed a partially folded, somewhat rigid structure before the trimerization step of protein biogenesis. The source of research support: University of Kentucky faculty start‐up fund to Wei Y. and Kentucky Opportunity Fellowship.