A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling.
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(+)/propidium(2+) 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
Theresia A
TA
Department of Pharmacology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1PD, UK.
van Veen
Hendrik W
HW
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002412:Cations; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011522:Protons
D001426:Bacterial Proteins; D002412:Cations; D008024:Ligands; C096807:LmrP protein, Lactococcus lactis; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011522:Protons