A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling

The FASEB JournalVolume 24, Issue 10 p. 3653-3661 Research CommunicationFree to Read A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling Theresia A. Schaedler, Theresia A. Schaedler Department of Pharmacology, University of Cambridge, Cambridge, UKSearch for more papers by this authorHendrik W. Van Veen, Corresponding Author Hendrik W. Van Veen hwv20@cam.ac.uk Department of Pharmacology, University of Cambridge, Cambridge, UK Correspondence: Department of Pharmacology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1PD, UK. E-mail: hwv20@cam.ac.ukSearch for more papers by this author Theresia A. Schaedler, Theresia A. Schaedler Department of Pharmacology, University of Cambridge, Cambridge, UKSearch for more papers by this authorHendrik W. Van Veen, Corresponding Author Hendrik W. Van Veen hwv20@cam.ac.uk Department of Pharmacology, University of Cambridge, Cambridge, UK Correspondence: Department of Pharmacology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1PD, UK. E-mail: hwv20@cam.ac.ukSearch for more papers by this author First published: 14 May 2010 https://doi.org/10.1096/fj.10-156927Citations: 4Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat ABSTRACT The multidrug major facilitator super-family 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 Citing Literature Volume24, Issue10October 2010Pages 3653-3661 RelatedInformation