Abstract
Secondary-active multidrug transporters can confer resistance on cells to pharmaceuticals by mediating their extrusion away from intracellular targets via substrate/H+(Na+) antiport. While the interactions of catalytic carboxylates in these transporters with coupling ions and substrates (drugs) have been studied in some detail, the functional importance of basic residues has received much less attention. The only two basic residues R260 and K357 in transmembrane helices in the Major Facilitator Superfamily transporter LmrP from Lactococcus lactis are present on the outer surface of the protein, where they are exposed to the phospholipid head group region of the outer leaflet (R260) and inner leaflet (K357) of the cytoplasmic membrane. Although our observations on the proton-motive force dependence and kinetics of substrate transport, and substrate-dependent proton transport demonstrate that K357A and R260A mutants are affected in ethidium-proton and benzalkonium-proton antiport compared to wildtype LmrP, our findings suggest that R260 and K357 are not directly involved in the binding of substrates or the translocation of protons. Secondary-active multidrug transporters are thought to operate by a mechanism in which binding sites for substrates are alternately exposed to each face of the membrane. Disulfide crosslinking experiments were performed with a double cysteine mutant of LmrP that reports the substrate-stimulated transition from the outward-facing state to the inward-facing state with high substrate-binding affinity. In the experiments, the R260A and K357A mutations were found to influence the dynamics of these major protein conformations in the transport cycle, potentially by removing the interactions of R260 and K357 with phospholipids and/or other residues in LmrP. The R260A and K357A mutations therefore modify the maximum rate at which the transport cycle can operate and, as the transitions between conformational states are differently affected by components of the proton-motive force, the mutations also influence the energetics of transport.
Highlights
Multidrug resistance originating from active efflux of pharmaceuticals from cells is a major obstacle in the treatment of microbial infections and human cancers [1,2,3,4]
As the presence of basic residues in transmembrane helices (TMHs) of membrane proteins can act as potent determinants of membrane topology [25,26], we compared the topology of the C-terminal half of Wt and mutant LmrP proteins through studies on the accessibility of the intracellular C-terminal His6-tag to digestion with proteinase K in preparations of well-defined inside-out membrane vesicles (ISOVs) and right-side-out membrane vesicles (RSOVs)
This difference in Vmax was reflected in the proton transport activities during benzalkonium-proton exchange, which were low for the R260A mutant compared to Wt LmrP and the K357A mutant (Fig. 7)
Summary
Multidrug resistance originating from active efflux of pharmaceuticals from cells is a major obstacle in the treatment of microbial infections and human cancers [1,2,3,4]. Active drug extrusion is mediated by multidrug transporters, which recognize a wide variety of structurally unrelated compounds [5,6,7]. On the basis of bioenergetic and structural criteria, multidrug transporters can be divided into two major classes: (i) primary-active ABC transporters, which utilize the free energy of ATP binding/ hydrolysis to efflux toxic substrate, and (ii) secondary-active transporters, which mediate drug extrusion in a coupled exchange with H+ and/or Na+ ions [8]. The multidrug transporter LmrP from Lactococcus lactis is a secondary-active transporter that is a member of the widely occurring Major Facilitator Superfamily (MFS) [9]. Its drug efflux activity was first found in genetic screens based on its ability to confer resistance on Escherichia coli to high concentrations of a monovalent amphiphilic ethidium cation [10]. During active and facilitated transport, LmrP is thought to mediate the transbilayer movement of substrates via a rigid body motion of the N- and C-terminal halves with a concomitant alternating access of substrate-binding sites to the inside surface and outside surface of the cytoplasmic membrane, in an analogous fashion as proposed for LacY, LeuT and other secondary-active transporters [14,15,16,17]
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