Abstract
The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.
Highlights
The ability of microbes to develop resistance to cytotoxic drugs, and to adapt rapidly to changes in the exposure to these compounds is an extremely important medical problem[1]
Multidrug transporters have been reinvented on multiple occasions in the course of evolution, and are found in six different protein families: the ATP-binding cassette (ABC) superfamily, resistance-nodulation-cell division (RND) family, multiple antibiotics and toxin extrusion (MATE) family, small multidrug resistance (SMR) family, proteobacterial antimicrobial compound efflux (PACE) family, and major facilitator superfamily (MFS)[3,4]
ATP-depleted cells were first pre-equilibrated with 2 μM propidium or ethidium, after which the efflux reaction was initiated with the addition of 25 mM glucose in the absence and presence of valinomycin and/or nigericin
Summary
The ability of microbes to develop resistance to cytotoxic drugs, and to adapt rapidly to changes in the exposure to these compounds is an extremely important medical problem[1]. In an inward-facing three-dimensional homology model of LmrP11 based on the crystal structure of the glycerol-phosphate/phosphate antiporter GlpT12, the N- and C-terminal membrane domains are suggested to form a large interior chamber containing three carboxyl residues (Asp-142, Asp-235 and Glu-327) that are surrounded by polar and aromatic residues. These carboxyl residues are predicted to be organised into two catalytic clusters, one containing Asp-235 and Glu-327 in the C-terminal domain (Cluster 1) with a distance of ~4.7 Å between the side chain centres and with a known ability to coordinate Ca2+ 13, and the other containing Asp-142 in the N-terminal domain (Cluster 2) at a distance of ~16.7 Å from Asp-235 and ~16.1 Å from Glu-327 (Fig. 1A). Our data highlight a flexibility in the location of catalytic carboxylates in this multidrug transporter
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