Switch Loop Flexibility Affects Substrate Transport of the AcrB Efflux Pump.

Area of Science:

• Structural biology
• Molecular biology
• Biochemistry

Background:

• The trimeric multidrug transporter AcrB is crucial for bacterial multidrug resistance.
• A functionally important switch loop within AcrB regulates substrate access to binding pockets.
• This loop's flexibility, suggested by glycine residues, is vital for transporter activity.

Purpose of the Study:

• To investigate the role of the switch loop's glycine residues in AcrB transporter function.
• To determine how altering loop flexibility impacts substrate transport.
• To elucidate the structural mechanisms underlying AcrB-mediated drug efflux.

Main Methods:

• Combinatorial substitution of glycine residues within the switch loop to proline.
• Structural and functional analyses of mutant AcrB transporters.
• Assessment of drug transport activity and loop backbone flexibility.

Main Results:

• Proline substitutions in the switch loop induced functional and structural asymmetry.
• Substitution on the PC1-proximal side abolished transport by reducing loop flexibility and restricting the pathway.
• Adjacent phenylalanine residues contribute to pathway blockage in rigidified loops, with their removal restoring transport.

Conclusions:

• Switch loop flexibility is essential for AcrB-mediated multidrug transport.
• Specific residue substitutions can disrupt transporter function by altering loop conformation and dynamics.
• Targeting loop flexibility offers a potential strategy for overcoming multidrug resistance.