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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 22, 2005 as doi:10.1096/fj.04-3558fje. |
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,1
* Department of Pharmacology and
Department of Biochemistry, University of Cambridge, Cambridge, UK
2 Correspondence: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK. E-mail: hwv20{at}cam.ac.uk
SPECIFIC AIMS
LmrA from Lactococcus lactis is a homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). Earlier work focused on the role of a carboxylate (E314) in proton-dependent ethidium transport by LmrA. The functional importance of this residue was suggested by its conservation in a wider family of related ABC transporters, but the structural basis of its role was not apparent. Here, we used homology modeling and functional analyses of LmrA mutants to define the structural environment of this key residue.
PRINCIPAL FINDINGS
1. Structural environment of E314
The crystal structure of MsbA from Vibrio cholera was used as a template to model LmrA-MD. The final model of LmrA-MD superimposes very well with the template, and all secondary structure elements are conserved, though they may vary in length. The root mean squared deviation between equivalent C
atoms in the superimposed structures is 0.57 Å, which reflects the excellent quality of the model. LmrA-MD contains six extended transmembrane helices per monomer with different orientations with respect to the lipid bilayer normal. The extracellular loops (ECLs) are short, ranging from 3 residues in ECL2 to 9 residues in ECL1. The intracellular portions connecting transmembrane helices 2 and 3, and 4 and 5, and the linker region between transmembrane helix 6 and the NBD, together form the so-called intracellular domain (ICD) and are predominantly -helical with the exception of a long extended region in ICD3.
Given the evidence supporting the LmrA-mediated transport of cationic amphiphilic substrates from the inner leaflet of the phospholipid bilayer, we searched for acidic residues in LmrA-MD with a favorable orientation to interact with cationic drugs from the inner membrane leaflet. The transmembrane helices of LmrA-MD form a principal groove that is proposed to be the site of substrate binding in MsbA. At the center of this groove in LmrA-MD, between the TM6 and its helical extension into ICD3, we have identified E314 as the first residue of an -helix at the beginning of ICD3 (Fig. 1
A, B) in a hydrophobic micro-environment formed by L107 in ICD1, and F311 and L315 in ICD3 (Fig. 1C
). E314 is conserved in MsbA from V. cholera, and the N- and C-terminal halves of human and murine P-glycoprotein. The residue is also present in the N-terminal, but not the C-terminal half of the human bile salt export pump (BSEP, also termed ABCB11), and in HorA from Lactobacillus brevis (Fig. 1D
). As E314 is located close to the interface between the lipid bilayer and the cytoplasm, it is in a favorable position to intercept the cationic moiety of a drug intercalated in the inner leaflet of the membrane (Fig. 1B
). E314 is also in a favorable position to play a role in the conformational coupling between the ATP binding/hydrolysis at the NBD and substrate movement by the MD in the same half-transporter, as TM6 is connected to the ATP-hydrolyzing NBD via the ICD3 region. In addition, both in the crystal structures of dimeric V. cholera MsbA and Salmonella typhimurium MsbA, and in a V. cholera MsbA-based model for human P-glycoprotein, the ICD3 region of one half-transporter is intimately packed to the NBD of the other half-transporter through its terminal extended coil. This suggests that any conformational change in the NBD region involving ATP binding and/or hydrolysis could be easily transmitted to E314 to produce downstream effects.
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2. Mutations of E314 in LmrA affect ethidium transport in intact cells
We analyzed the role of E314 in ethidium transport by comparing wild-type LmrA with mutants in which E314 was substituted by the shorter aspartic acid (E314D) or neutral alanine (E314A). Both substitutions strongly inhibited facilitated ethidium influx in ATP-depleted cells, and caused a significant reduction in the Vmax of facilitated ethidium efflux rather than a change in the Kt for ethidium, in agreement with a role of E314 in the catalytic mechanism of LmrA rather in direct binding of the substrate. At pH 7.0, active ethidium efflux by the E314D mutant LmrA (in the presence of glucose as a source of metabolic energy) was similar to that of the E314A mutant, but strongly reduced compared with the activity of the wild-type protein. At pH 8.0, active ethidium efflux by the E314D mutant LmrA was dramatically enhanced compared with the E314A mutant, and found to be similar to the efflux mediated by the wild-type protein. Hence, the transport activity of the E314D mutant could be rescued at pH 8.0, implying that carboxylates at this position exhibit a relatively high pKa value compared with the pKa observed for aspartic acid and glutamic acid isolated in solution. This observation may relate to the presence of residue 314 in a hydrophobic microenvironment (Fig. 1C
), which may inhibit the deprotonation of the carboxyl moiety. Similar to E314 in LmrA, the reported pKa of many of the carboxylates responsible for proton translocation by secondary active transporters and primary proton pumps is unusually high. Coupling of the biological activities of these proteins to metabolic energy has been suggested to modify the pKa of the carboxylates, resulting in their protonation or deprotonation followed by conformational changes enabling vectorial proton translocation.
3. Ethidium/proton cotransport in proteoliposomes
Recent work showed that LmrA-MD is able to mediate the uptake of ethidium in cells in cotransport with protons in a reaction driven by the transmembrane potential (
, interior negative) and the transmembrane chemical proton gradient (pH, interior alkaline). To compare the dependence on the protonmotive-force by wild-type LmrA-MD and E314A mutant LmrA-MD, the proteins were functionally reconstituted in an inside-out fashion into DNA-loaded proteoliposomes in which a –ZpH (interior alkaline) of –60 mV was imposed artificially through the 100-fold dilution of acetate-loaded proteoliposomes into an acetate-free buffer. While LmrA-MD containing proteoliposomes exhibited a significant uptake of ethidium under these conditions, no uptake of ethidium was observed in E314A mutant LmrA-MD-containing proteoliposomes above that obtained in control liposomes (Fig. 2
A). As the pH was imposed by increasing the lumen pH of the (proteo)liposomes rather than decreasing the pH of the external environment, where E314 resides, these data show the importance of the transmembrane pH gradient in E314-dependent ethidium transport by LmrA-MD. Upon the imposition of a (interior negative) of –120 mV in proteoliposomes the activities of E314A mutant LmrA-MD and LmrA-MD were comparable, demonstrating the functionality of E314A mutant LmrA-MD in these experiments (Fig. 2B
). No uptake above the equilibration level was observed in the proteoliposomes in the absence of an imposed gradient (Fig. 2C
).
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CONCLUSIONS AND SIGNIFICANCE
In conclusion, we used homology modeling to determine the structural basis of the role of E314 in proton-coupled ethidium transport by LmrA. The residue is present in a hydrophobic micro-environment that probably elevates its pKa, accounting for the pH dependency of drug efflux we observe. Functional analyses of wild-type and mutant proteins in cells and proteoliposomes support our proposal for the mechanistic role of E314 in proton-coupled drug transport. Our findings show that in the absence of ATP binding/hydrolysis, the conformational changes in LmrA-MD required for ethidium transport can be initiated by the imposition of a pH, for protein containing E314. As E314 is localized in ICD3, a segment in the intracellular domain of LmrA connecting the MD to the NBD, these findings imply a putative new role of carboxylate-dependent proton transport in energy transduction in this ABC transporter. Carboxylate-dependent proton transport by LmrA and related proteins could arise from 1) the transport cycle-dependent alternate exposure of carboxylates to hydrophobic and hydrophilic environments, where these carboxylates bind and release protons, respectively, 2) the transport cycle-dependent transition of carboxylates between a state where the anionic form is stabilized by interactions with polar or cationic residues, and a state in which the anionic form can be protonated, and 3) association/dissociation reactions in which cationic drugs and protons compete for binding to isolated carboxylates in a hydrophobic environment. Finally, the ability of LmrA to transport protons might relate to a physiological activity of this transporter in L. lactis, which has yet to be identified.
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FOOTNOTES
1 Current address: Ce.S.I. Centro Studi sull’Invecchiamento e Dipartimento di Scienze Biomediche, Universita’ di Chieti "G. D’Annunzio," Via dei Vestini 31, 66013 Chieti, Italy. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3558fje;
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