18-Crown-6-ether Utilizes Distinct Allosteric Interactions to Uncouple Transport by the Multidrug Efflux Pump EmrE

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• The multidrug efflux pump EmrE from Escherichia coli exhibits multiple transport functions, presenting a potential therapeutic target.
• Small-molecule substrates can induce transporter-dependent susceptibility, contrasting with the typical antibiotic resistance phenotype.

Purpose of the Study:

• To investigate the binding site and mechanism of action of the small-molecule substrate 18-crown-6-ether on the EmrE transporter.
• To elucidate the role of lysine 22 and threonine 56 in the allosteric regulation of EmrE transport.

Main Methods:

• Nuclear Magnetic Resonance (NMR) chemical shift perturbations were used to identify the binding site of 18-crown-6-ether.
• In vivo EC50 assays and liposomal leak assays were employed to assess transporter activity and substrate-induced effects.

Main Results:

• 18-crown-6-ether was confirmed to bind to lysine 22 (K22) of the EmrE transporter.
• This substrate was shown to trigger uncoupled proton leak, indicating a disruption of normal transport regulation.
• Mutational analysis of K22 further solidified the importance of this residue and T56 in allosteric regulation.

Conclusions:

• Lysine 22 is a critical residue for the allosteric regulation of EmrE, particularly in interaction with the C-terminal tail.
• Understanding EmrE's transport mechanism and regulation by susceptibility substrates offers insights applicable to other multidrug-resistant efflux pumps.