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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.

Multidrug resistance of pathogenic microorganisms and mammalian tumors can be associated with the overexpression of multidrug transporters. These integral membrane proteins are capable of extruding a wide range of structurally unrelated compounds from the cell. Among the different classes of multidrug transporters are the ATP binding cassette (ABC) transporters, which are dependent on the binding and hydrolysis of ATP. In the past five years, many researchers have built homology models of ABC extrusion systems using the atomic coordinates of crystallized MsbA, a lipopolysaccharide transporter in Gram-negative bacteria. Likewise, we have previously used the Vibrio cholera MsbA structure as a template in the modeling of the multidrug transporter LmrA from Lactococcus lactis. In view of the recently discovered inaccuracies in the MsbA structure, we have remodelled LmrA using the atomic coordinates of the MsbA homologue Sav1866 from Staphylococcus aureus. To compare and test our MsbA-based and Sav1866-based LmrA models we performed cysteine cross-linking at three key positions in LmrA. The pattern of cross-linking at these positions was consistent with the overall topology of transmembrane helices in Sav1866, suggesting that its crystal structure might be physiologically relevant. We recently identified E314 as a residue important in proton conduction by LmrA. The predicted location of this residue at the interface between the two half-transporters in the Sav1866-based homodimer, within the inner leaflet of the phospholipid bilayer, provides a new structural basis for the role of E314 in LmrA-mediated transport.

ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

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.

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.

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis , our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis . MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K i values of 16 and 11 μM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K i of 57 μM, in a similar range as the K i for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).

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.