Dissecting the function of a protruding loop in AcrB trimerization

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• AcrB from E. coli is a key multidrug transporter belonging to the resistance-nodulation-cell division (RND) family.
• The AcrB homotrimer structure involves significant inter-subunit interactions mediated by a protruding loop and a binding tunnel.

Purpose of the Study:

• To investigate the precise role of the protruding loop in AcrB trimerization and function.
• To identify critical residues and interaction dynamics within the loop-tunnel interface.

Main Methods:

• Alanine scanning mutagenesis of the loop region in AcrB.
• Assessing the impact of mutations on AcrB protein activity and stability.
• Construction and analysis of a chimeric protein using the MexB loop.

Main Results:

• Not all conserved residues within the loop are essential for AcrB function; critical residues are not always conserved.
• A chimeric AcrB protein with the MexB loop retained partial activity and existed as a mix of monomer and trimer.
• Mutations causing reduced function clustered in specific areas, suggesting a potential inter-subunit recognition or stabilization site.

Conclusions:

• The loop-tunnel interaction is vital for AcrB trimerization and efflux but demonstrates considerable flexibility and tolerance to mutations.
• Specific regions within the loop may act as key sites for subunit recognition and stabilization during trimer formation.