title: Folding of AcrB Subunit Precedes Trimerization.
abstract: 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, AcrB(Δloop), was created through the truncation of a protruding loop that appeared to contribute to the stability of an AcrB trimer. AcrB(Δloop) expressed at a level similar to that of wild-type AcrB. The secondary structure content and tertiary conformation of AcrB(Δloop) were very similar to those of wild-type AcrB. However, when expressed in an acrB-deficient strain, AcrB(Δloop) failed to complement its defect in drug efflux. Results from blue native polyacrylamide gel electrophoresis and chemical cross-linking experiments suggested that AcrB(Δloop) 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. AcrB(Δloop) 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.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.forename: Meng
authors.initials: M
authors.lastname: Zhong
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; C083496:AcrB protein, E coli