Journal
FASEB JOURNAL
Volume 24, Issue 10, Pages 3653-3661Publisher
FEDERATION AMER SOC EXP BIOL
DOI: 10.1096/fj.10-156927
Keywords
multidrug resistance; secondary-active transport; ligand binding; coupling stoichiometry
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Funding
- Cambridge European Trusts
- Pembroke College
- Kurt Hahn Trust
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The multidrug major facilitator superfamily transporter LmrP from Lactococcus lactis mediates protonmotive-force dependent efflux of amphiphilic ligands from the cell. We compared the role of membrane-embedded carboxylates in transport and binding of divalent cationic propidium and monovalent cationic ethidium. D235N, E327Q, and D142N replacements each resulted in loss of electrogenicity in the propidium efflux reaction, pointing to electrogenic 3H(+)/propidium2(+) antiport. During ethidium efflux, single D142N and D235N replacements resulted in apparent loss of electrogenicity, whereas the E327Q substitution did not affect the energetics, consistent with electrogenic 2H(+)/ethidium(+) antiport. Different roles of carboxylates were also observed in fluorescence anisotropy-based ligand-binding assays. Whereas D235 and E327 were both involved in propidium binding, the loss of one of these carboxylates could be compensated for by the other in ethidium binding. The D142N replacement did not affect the binding of either ligand. These data point to the presence of a dedicated proton binding site containing D142, and a flexible proton/ligand binding site containing D235 and E327, the contributions to proton and ligand binding of which depend on the chemical structure of the bound ligand. Our findings provide the first evidence that multidrug transport by secondary-active transporters can be associated with variable ion coupling.-Schaedler, T. A., van Veen, H. W. A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling. FASEB J. 24, 3653-3661(2010). www.fasebj.org
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