ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter.

The ABC multidrug exporter MsbA mediates the translocation of lipopolysaccharides and phospholipids across the plasma membrane in Gram-negative bacteria. Although MsbA is structurally well characterised, the energetic requirements of lipid transport remain unknown. Here, we report that, similar to the transport of small-molecule antibiotics and cytotoxic agents, the flopping of physiologically relevant long-acyl-chain 1,2-dioleoyl (C18)-phosphatidylethanolamine in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and the chemical proton gradient as sources of metabolic energy. In contrast, the flopping of the large hexa-acylated (C12-C14) Lipid-A anchor of lipopolysaccharides is only ATP dependent. This study demonstrates that the energetics of lipid transport by MsbA is lipid dependent. As our mutational analyses indicate lipid and drug transport via the central binding chamber in MsbA, the lipid availability in the membrane can affect the drug transport activity and vice versa.

Researchers study how different forms of metabolic energy are coupled to drug extrusion by an ATP-binding cassette transporter.

The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues colocalise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.The ability of microbes to develop resistance to cytotoxic drugs, and to adapt rapidly to changes in the exposure to these compounds is an extremely important medical problem 1 . Drug resistance can be specific for a single drug or class of drugs, or occur simultaneously for a wide variety of toxic compounds that are structurally and functionally unrelated. The latter phenomenon is called multidrug resistance, and is recognised as an important mechanism of bacterial resistance to valuable clinical antibiotics. In many cases, the development of multidrug resistance is due to the enhanced expression levels of drug pumps in the cell 2 . These pumps mediate the extrusion of antimicrobials across the cell envelope, away from intracellular targets. Enzyme-ligand interactions are usually based on very specific interactions that allow discrimination between enantiomers of the same ligand. The ability of multidrug transporters to recognise a broad spectrum of structurally unrelated drugs is intriguing.Multidrug transporters have been reinvented on multiple occasions in the course of evolution, and are found in six different protein families: the ATP-binding cassette (ABC) superfamily, resistance-nodulation-cell division (RND) family, multiple antibiotics and toxin extrusion (MATE) family, small multidrug resistance (SMR) family, proteobacterial antimicrobial compound efflux (PACE) family, and major facilitator superfamily (MFS) 3,4 . It is generally accepted that members of the ABC family couple drug efflux to ATP binding and hydrolysis, whereas the members of the other 5 families operate by a secon...

Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals. Our understanding of how MATE transporters work, has mostly relied on protein structures and MD simulations. However, the energetics of drug transport has not been studied in detail. Many MATE transporters utilise the electrochemical H+ or Na+ gradient to drive substrate efflux, but NorM-VC from Vibrio cholerae can utilise both forms of metabolic energy. To dissect the localisation and organisation of H+ and Na+ translocation pathways in NorM-VC we engineered chimaeric proteins in which the N-lobe of H+-coupled NorM-PS from Pseudomonas stutzeri is fused to the C-lobe of NorM-VC, and vice versa. Our findings in drug binding and transport experiments with chimaeric, mutant and wildtype transporters highlight the versatile nature of energy coupling in NorM-VC, which enables adaptation to fluctuating salinity levels in the natural habitat of V. cholerae.