ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled.

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.

Effects of nucleotide binding to LmrA: A combined MAS-NMR and solution NMR study.

ABC transporters are fascinating examples of fine-tuned molecular machines that use the energy from ATP hydrolysis to translocate a multitude of substrates across biological membranes. While structural details have emerged on many members of this large protein superfamily, a number of functional details are still under debate. High resolution structures yield valuable insights into protein function, but it is the combination of structural, functional and dynamic insights that facilitates a complete understanding of the workings of their complex molecular mechanisms.

Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.

ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes.

Structural and functional conservation of key domains in InsP3 and ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca(2+) channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP(3)R gating is initiated by InsP(3) binding to the InsP(3)-binding core (IBC, residues 224-604 of InsP(3)R1) and it requires the suppressor domain (SD, residues 1-223 of InsP(3)R1).

IP3 receptors: some lessons from DT40 cells.

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that are regulated by IP3 and Ca2+ and are modulated by many additional signals. These properties allow them to initiate and, via Ca2+-induced Ca2+ release, regeneratively propagate Ca2+ signals evoked by receptors that stimulate formation of IP3. The ubiquitous expression of IP3R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP3R.

A multidrug ABC transporter with a taste for salt.

Background: LmrA 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.

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP.

Caught in the act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR.

The ATP binding cassette (ABC) transporter LmrA from Lactococcus lactis transports cytotoxic molecules at the expense of ATP. Molecular and kinetic details of LmrA can be assessed by solid-state nuclear magnetic resonance (ssNMR), if functional reconstitution at a high protein-lipid ratio can be achieved and the kinetic rate constants are small enough. In order to follow ATP hydrolysis directly by 31P-magic angle spinning (MAS) nuclear magnetic resonance (NMR), we generated such conditions by reconstituting LmrA-dK388, a mutant with slower ATP turnover rate, at a protein-lipid ration of 1:150.

Functional role of transmembrane helix 6 in drug binding and transport by the ABC transporter MsbA.

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).

Multidrug transport by the ABC transporter Sav1866 from Staphylococcus aureus.

Sav1866 is an ATP-binding cassette (ABC) protein from the pathogen Staphylococcus aureus and is a homologue of bacterial and human multidrug ABC transporters. Recently, the three-dimensional crystal structure of Sav1866 was determined at 3.0 A resolution [Dawson, R.

A functional steroid-binding element in an ATP-binding cassette multidrug transporter.

The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid interactions in ABC transporters are unknown.

On the energy-dependence of Hoechst 33342 transport by the ABC transporter LmrA.

LmrA is an ATP-binding cassette (ABC) multidrug transporter from Lactococcus lactis, and is a structural homologue of the human multidrug resistance P-glycoprotein (ABCB1), the overexpression of which is associated with multidrug resistance in tumours. We recently observed that a truncated version of LmrA lacking the nucleotide-binding domain mediates a proton motive force-dependent ethidium transport reaction by catalyzing proton-ethidium symport. This finding raised the question whether proton motive force-dependent transport can also be observed for other drugs, and whether this reaction is also relevant for full-length LmrA.

ABCG transporters: structure, substrate specificities and physiological roles : a brief overview.

The ATP-binding cassette (ABC) transporter superfamily is one of the largest protein families with representatives in all kingdoms of life. Members of this superfamily are involved in a wide variety of transport processes with substrates ranging from small ions to relatively large polypeptides and polysaccharides. The G subfamily of ABC transporters consists of half-transporters, which oligomerise to form the functional transporter.

New structure model for the ATP-binding cassette multidrug transporter LmrA.

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.

Two proton translocation pathways in a secondary active multidrug transporter.

LmrP is a secondary active multidrug transporter from Lactococcus lactis. The protein belongs to the major facilitator superfamily and utilizes the electrochemical proton gradient (inside negative and alkaline) to extrude a wide range of lipophilic cations from the cell. Previous work has indicated that ethidium, a monovalent cationic substrate, is exported by LmrP by electrogenic antiport with two (or more) protons.

New light on multidrug binding by an ATP-binding-cassette transporter.

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.

Drug-lipid A interactions on the Escherichia coli ABC transporter MsbA.

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.

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.

An ABC transporter with a secondary-active multidrug translocator domain.

Multidrug resistance, by which cells become resistant to multiple unrelated pharmaceuticals, is due to the extrusion of drugs from the cell's interior by active transporters such as the human multidrug resistance P-glycoprotein. Two major classes of transporters mediate this extrusion. Primary-active transporters are dependent on ATP hydrolysis, whereas secondary-active transporters are driven by electrochemical ion gradients that exist across the plasma membrane.