Switch Loop Flexibility Affects Substrate Transport of the AcrB Efflux Pump

摘要

Abstract The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity. Graphical abstract Display Omitted Highlights ? We substituted glycine residues to proline in the AcrB switch loop. ? We observed an asymmetrical substitution sensitivity on drug transport. ? Substitutions affecting activity show switch loop becoming more rigid. ? Flexibility reduction narrows the drug transport path, caused by two Phe residues. ? Removal of the phenyl rings in the rigid loop restores transport activity.