Premium
A critical role of a carboxylate in proton conduction by the ATP‐binding cassette multidrug transporter LmrA
Author(s) -
Shilling Richard,
Federici Luca,
Walas Fabien,
Venter Henrietta,
Velamakanni Saroj,
Woebking Barbara,
Balakrishnan Lekshmy,
Luisi Ben,
Veen Hendrik W.
Publication year - 2005
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.04-3558fje
Subject(s) - atp binding cassette transporter , efflux , transporter , lactococcus lactis , biochemistry , biology , mutant , multiple drug resistance , chemistry , bacteria , genetics , gene , lactic acid , antibiotics
ABSTRACT The ATP binding cassette (ABC) transporter LmrA from the bacterium Lactococcus lactis is a homolog of the human multidrug resistance P‐glycoprotein (ABCB1), the activity of which impairs the efficacy of chemotherapy. In a previous study, LmrA was shown to mediate ethidium efflux by an ATP‐dependent proton‐ethidium symport reaction in which the carboxylate E314 is critical. The functional importance of this key residue for ABC proteins was suggested by its conservation in a wider family of related transporters; however, the structural basis of its role was not apparent. Here, we have used homology modeling to define the structural environment of E314. The residue is nested in a hydrophobic environment that probably elevates its p K a , accounting for the pH dependency of drug efflux that we report in this work. Functional analyses of wild‐type and mutant proteins in cells and proteoliposomes support our proposal for the mechanistic role of E314 in proton‐coupled ethidium transport. As the carboxylate is known to participate in proton translocation by secondary‐active transporters, our observations suggest that this substituent can play a similar role in the activity of ABC transporters.