Mechanistic insights into the secondary-active multidrug transporter LmrP

Abstract

Antibiotic resistance is a global crisis and one of the important contributing mechanisms is active drug extrusion by bacterial multidrug transporters. LmrP is a Major Facilitator Superfamily (MFS) multidrug transporter which, when overexpressed in bacteria, can confer resistance to 22 clinically important antibiotics. The structural data for MFS proteins support an alternating access mechanism, in which these transporters alternate between two structural states that enable the binding of drugs on one side of the membrane and release on the other side. In LmrP, this mechanism is driven by the membrane potential and chemical proton gradient that exist across the plasma membrane as the two components of the proton motive force. We have previously demonstrated the role of catalytic carboxylates in LmrP in proton coupling. In this PhD project, the role of these carboxylates in regulating the key conformational changes in LmrP was determined in the native plasma membrane. For this purpose, the orientation of the substrate-binding chamber of wildtype LmrP and mutants (LmrP-D68N, LmrP-D142N, LmrP-D235N and LmrP-E327Q) was assessed in membrane vesicles in cysteine accessibility assays. The results show that D68 and D142 in the N-terminal half, and D235 and E327 in the C-terminal half, play a vital role in the conformational transitions between the outward-open and inward-open conformations, and that, within each half, the carboxylates contribute in a similar phenotypic fashion. In particular, the deprotonation of D235 and E327 stabilises an inward-open conformation, whereas the protonation of these residues leads to an outward-open conformation. For D68 and D142 these responses are opposite to those of E327 and D235. In these experiments, the wildtype protein appears to be inaccessible to either side of the membrane suggesting it adopts an occluded conformation in the absence of the proton motive force. These results are interpreted in a structural context, and a mechanistic model is presented for the transport of divalent cationic propidium and monovalent cationic ethidium. I also established an antibiotic binding assay to assess conformational changes in LmrP. The results show that the binding affinity for erythromycin is higher in the inward-open state and that two antibiotic molecules can bind in the drug binding chamber with different affinities. Finally, I established that LmrP can transport phospholipids, which raises interesting questions about the role of lipids in antibiotic transport by this efflux pump. The information gained in this PhD research contributes to our knowledge of the molecular mechanisms of multidrug transporters and might ultimately lead to strategies that can combat antimicrobial drug resistance.