A critical role of a carboxylate in proton conduction by the ATP-binding cassette multidrug transporter LmrA.
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 pKa, 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.
Shilling
Richard
R
Department of Pharmacology, University of Cambridge, Cambridge, UK.
Federici
Luca
L
Walas
Fabien
F
Venter
Henrietta
H
Velamakanni
Saroj
S
Woebking
Barbara
B
Balakrishnan
Lekshmy
L
Luisi
Ben
B
van Veen
Hendrik W
HW
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport; D002264:Carboxylic Acids / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D030562:Databases, Protein; D004352:Drug Resistance, Microbial; D004996:Ethidium / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011522:Protons; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D002264:Carboxylic Acids; D004996:Ethidium; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011522:Protons; C101824:LmrA protein, Lactococcus lactis