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The ATP-binding cassette transporter MsbA in Gram-negative bacteria can transport antibiotics and toxic ions. However, the key functional regions in MsbA which determine substrate specificity remain to be identified. We recently examined published mutations in the human MsbA homologue ABCB1 that alter multidrug transport in cells and identified mutations that affect the specificity for individual substrates (termed change-in-specificity mutations). When superimposed on the corrected 3.7 A resolution crystal structure of homodimeric MsbA from S almonella typhimurium, these change-in-specificity mutations colocalize in a major groove in each of the two "wings" of transmembrane helices (TMHs) that point away from one another toward the periplasm. Near the apex of the groove, the periplasmic side of TMH 6 in both monomers contains a hotspot of change-in-specificity mutations and residues which, when replaced with cysteines in ABCB1, covalently interact with thiol-reactive drug analogues. We tested the importance of this region of TMH 6 for drug-protein interactions in Escherichia coli MsbA. In particular, we focused on conserved S289 and S290 residues in the hotspot. Their simultaneous replacement with alanine (termed the SASA mutant) significantly reduced the level of binding and transport of ethidium and Taxol by MsbA, whereas the interactions with Hoechst 33342 and erythromycin remained unaffected. Hence, the SASA mutation is associated with a change-in-specificity phenotype analogous to that of the change-in-specificity mutations in ABCB1. This study demonstrates for the first time the significance of TMH 6 for drug binding and transport by MsbA. Based on these data, a possible mechanism for alternating access of drug-binding surfaces in MsbA is discussed.

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BackgroundLmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H+-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known.Methodology/Principal FindingsWe examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H+-Na+-Cl− symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift.Conclusions/SignificanceThe observations on H+-Na+-Cl− co-transport substantiate earlier suggestions of H+-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter.

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.

Recent crystal structures of the multidrug ATP-binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification. The structures of homodimeric MsbA reveal a pair of glutamate residues (E208 and E208') in the intracellular domains of its two half-transporters, close to the nucleotide-binding domains (NBDs), which are in close proximity of each other in the outward-facing state but not in the inward-facing state. Using intermolecular cysteine crosslinking between E208C and E208C' in E. coli MsbA, we demonstrate that the NBDs dissociate in nucleotide-free conditions and come close on ATP binding and ADP·vanadate trapping. Interestingly, ADP alone separates the half-transporters like a nucleotide-free state, presumably for the following catalytic cycle. Our data fill persistent gaps in current studies on the conformational dynamics of a variety of ABC exporters. Based on a single biochemical method, the findings describe a conformational cycle for a single ABC exporter at major checkpoints of the ATPase reaction under experimental conditions, where the exporter is transport active.

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The movement of drugs across biological membranes is mediated by two major classes of membrane transporters. Primary-active, ABC (ATP-binding cassette) multidrug transporters are dependent on ATP-binding/hydrolysis, whereas secondary-active multidrug transporters are coupled to the proton (or sodium)-motive force that exists across the plasma membrane. Recent work on LmrA, an ABC multidrug transporter in Lactococcus lactis, suggests that primary- and secondary-active multidrug transporters share functional and structural features. Some of these similarities and their implications for the mechanism of transport by ABC multidrug transporters will be discussed.