Biophysical characterization of E. coli TolC interaction with the known blocker hexaamminecobalt

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 11; pp. 2702 - 2709
• Main Authors: Gilardi, A., Bhamidimarri, S.P., Brönstrup, M., Bilitewski, U., Marreddy, R.K.R., Pos, K.M., Benier, L., Gribbon, P., Winterhalter, M., Windshügel, B.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2017
• Subjects: antibacterial properties; antibiotic resistance; Bacterial Outer Membrane Proteins - antagonists & inhibitors; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - drug effects; Bacterial Outer Membrane Proteins - genetics; Biophysical Phenomena; cations; Cobalt - pharmacology; dissociation; Drug Resistance, Multiple, Bacterial - drug effects; Drug Resistance, Multiple, Bacterial - genetics; drugs; electric power; Electrophysiology; erythromycin; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli Proteins - antagonists & inhibitors; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Gene Expression Regulation, Bacterial - drug effects; Hexaamminecobalt; ligands; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; minimum inhibitory concentration; outer membrane proteins; Surface Plasmon Resonance; TolC; transporters; CCCP; Hexaamminecobalt; SPR; DMSO; TolC; AMR; MIC; Electrophysiology; RND; OMF; Surface plasmon resonance; HC
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2017.07.014

The tripartite efflux pump AcrAB-TolC in E. coli is involved in drug resistance by transporting antibiotics out of the cell. The outer membrane protein TolC can be blocked by various cations, including hexaamminecobalt, thereby TolC represents a potential target for reducing antimicrobial resistance as its blockage may improve efficacy of antibiotics. We utilized single channel electrophysiology measurements for studying TolC conductance in the absence and presence of the known TolC blocker hexaamminecobalt. Association and dissociation constants of hexaamminecobalt were determined using surface plasmon resonance measurements. Minimum inhibitory concentration (MIC) assays in the absence and presence of antibiotics were carried out for investigating the antibacterial effect of hexaamminecobalt and its potential to reduce MICs. TolC gating in the absence of any ligand is voltage dependent and asymmetric at high applied voltages. Hexaamminecobalt binds to TolC with high affinity and kinetic data revealed fast association and dissociation rates. Despite potent binding to TolC, hexaamminecobalt does not possess an intrinsic antimicrobial activity against E. coli nor does it reduce MIC values of antibiotics erythromycin and fusidic acid. TolC opening can be effectively blocked by small molecules. More potent channel blockers are needed in order to investigate the eligibility of TolC as drug target. TolC, a potentially interesting pharmaceutical target can be addressed by small molecules, blocking the channel. Biophysical characterization of the binding processes will support future identification and optimisation of more potent TolC blockers in order to validate TolC as a pharmaceutical target. •Electrophysiology measurements at higher voltages reveal TolC gating to be voltage-dependent and asymmetric.•Hexaamminecobalt binding to TolC involves fast association and dissociation rates.•TolC opening is blocked by hexaamminecobalt in its closed and open conformation.•Hexaamminecobalt does not possess antimicrobial activity in E.coli strains.