Folding of AcrB Subunit Precedes Trimerization

Area of Science:

• Microbiology
• Structural Biology
• Protein Biochemistry

Background:

• AcrB is a key efflux pump in Gram-negative bacteria, crucial for antimicrobial resistance.
• The assembly mechanism of AcrB, a homo-trimer, is not fully understood, with two proposed pathways: a three-stage (folding then association) or a two-stage (simultaneous folding and association).

Purpose of the Study:

• To investigate the assembly pathway of AcrB by attempting to create a stable, folded monomeric mutant.
• To determine if folded monomers can exist independently, providing evidence for the three-stage assembly pathway.

Main Methods:

• Engineered a monomeric AcrB mutant (AcrB(Δloop)) by truncating a loop involved in trimer stability.
• Assessed protein expression, secondary and tertiary structure using spectroscopy.
• Evaluated drug efflux complementation in an acrB-deficient strain.
• Utilized blue native polyacrylamide gel electrophoresis and chemical cross-linking to determine oligomeric state.
• Tested for dominant-negative effects in a wild-type Escherichia coli strain.

Main Results:

• AcrB(Δloop) was expressed at levels comparable to wild-type AcrB, with similar secondary and tertiary structures.
• AcrB(Δloop) failed to restore drug efflux function in an acrB-deficient strain.
• Biochemical analyses indicated that AcrB(Δloop) exists as a monomer.
• The mutant did not significantly inhibit wild-type AcrB assembly, suggesting it cannot co-assemble.

Conclusions:

• AcrB(Δloop) represents the first reported monomeric mutant of the intrinsically trimeric AcrB.
• The existence and behavior of AcrB(Δloop) support a three-stage assembly pathway for AcrB, involving pre-folded monomers.
• This study provides critical insights into the fundamental mechanisms of bacterial efflux pump assembly and function.