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Basic residues R260 and K357 affect the conformational dynamics of the major facilitator superfamily multidrug transporter LmrP.

Published version
Peer-reviewed

Type

Article

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Authors

Wang, Wei 
van Veen, Hendrik W 

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.

Description

Keywords

Arginine, Bacterial Proteins, Benzalkonium Compounds, Benzimidazoles, Binding Sites, Biological Transport, Cell Membrane, Cross-Linking Reagents, Cysteine, Drug Resistance, Multiple, Ethidium, Hydrogen-Ion Concentration, Immunoblotting, Lactococcus lactis, Lysine, Membrane Transport Proteins, Mutagenesis, Site-Directed, Mutation, Protein Conformation, Proton-Motive Force, Protons

Journal Title

PLoS One

Conference Name

Journal ISSN

1932-6203
1932-6203

Volume Title

7

Publisher

Public Library of Science (PLoS)
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/I002383/1)
Biotechnology and Biological Sciences Research Council (BB/H00288X/1)