Mechanistic studies of a multidrug efflux protein from Escherichia coli

Abstract

Inappropriate use of antibiotics has created the opportunity for rapid rise in bacteria able to resistant a wide range of antimicrobial agents. These multidrug resistant (MDR) organisms pose a “catastrophic threat” to modern medical procedures which rely upon effective infection control. Therefore, there is great need to understand the mechanisms behind the MDR, including the efflux pumps which reduce cytoplasmic concentrations of antimicrobials to levels no longer toxic to the cell. One such membrane protein from Escherichia coli is MdtM, a 12 TM antiporter of the Major Facilitator Superfamily (MFS). MdtM, like other members of the MFS, utilises the energy stored in the electrochemical gradient to drive the extrusion of drugs from the cytoplasm. Therefore, protons take part in many critical interactions with MdtM. Chloramphenicol and tetraphenylphosphonium appear to compete with protons for binding to MdtM at pH values < 6.7. This apparent competition may be as a result of direct competition for the binding site but is more likely as a result of proton mediated conformational changes. These changes could lead to MdtM no longer being in the high affinity inward open conformation. Charged residues play a critical role in linking proton and substrate transport. Using in silico analysis of homology models, critical interactions between charged residues have been identified and functionally investigated. The conformational switch and charge relay triad have been identified. MdtM tolerates mutation of the conserved charged residue inside the binding site; the transport activity is reduced but not abolished and binding affinity is not impacted to the extent of removal of binding residues. Mutation to the conserved residues of loop regions has a detrimental impact on the stability of the transporter when extracted from the membrane.

Date of Award	Dec 2018
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Christopher Law (Supervisor)