Folding of AcrB Subunit Precedes Trimerization

AcrB and its homologues are major players in the efflux of anti-microbials out of Gram-negative bacteria. The structural and functional unit of AcrB is a homo-trimer. The assembly process of obligate membrane protein oligomers, including AcrB, remains elusive. It is not clear if an individual subunit folds into a monomeric form first followed by association (three-stage pathway) or if association occurs simultaneously with subunit folding (two-stage pathway). To answer this question, we investigated the feasibility of creating a folded monomeric AcrB mutant. The existence of well-folded monomers in the cell membrane would be an evidence of a three-stage pathway. A monomeric AcrB mutant, AcrBΔloop, was created through the truncation of a protruding loop that appeared to contribute to the stability of an AcrB trimer. AcrBΔloop expressed at a level similar to that of wild-type AcrB. The secondary structure content and tertiary conformation of AcrBΔloop were very similar to those of wild-type AcrB. However, when expressed in an acrB-deficient strain, AcrBΔloop failed to complement its defect in drug efflux. Results from blue native polyacrylamide gel electrophoresis and chemical cross-linking experiments suggested that AcrBΔloop existed as a monomer. The expression of this monomeric mutant in a wild-type Escherichia coli strain did not have a significant dominant-negative effect, suggesting that the mutant could not effectively co-assemble with genomic AcrB. AcrBΔloop is the first monomeric mutant reported for the intrinsically trimeric AcrB. The structural characterization results of this mutant suggest that the oligomerization of AcrB occurs through a three-stage pathway involving folded monomers. [Display omitted]