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The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.

The ATP-binding-cassette transporter OpuA from Lactococcus lactis is composed of two ATPase subunits (OpuAA) and two subunits (OpuABC) with the transmembrane domain fused to an extracellular substrate-binding protein. Of the almost 1900 homologues of OpuA known to date, a subset has an amino-terminal amphipathic helix (plus extra transmembrane segment) fused to the core of the transmembrane domain of the OpuABC subunit. FRET measurements indicate that the amphipathic alpha-helix is located close to the membrane surface, where its hydrophobic face interacts with the transport protein rather than the membrane lipids. Next, we determined the functional role of this accessory region by engineering the amphipathic alpha-helix. We analyzed the consequence of the mutations in intact cells by monitoring growth and transport of glycine betaine under normal and osmotic stress conditions. More detailed studies were performed in hybrid membrane vesicles, proteoliposomes, and bilayer nanodisks. We show that the amphipathic alpha-helix of OpuA is necessary for high activity of OpuA but is not critical for the biogenesis of the protein or the ionic regulation of transport.

The cystathionine beta-synthase module of OpuA in conjunction with an anionic membrane surface acts as a sensor of internal ionic strength, which allows the protein to respond to osmotic stress. We now show by chemical modification and cross-linking studies that CBS2-CBS2 interface residues are critical for transport activity and/or ionic regulation of transport, whereas CBS1 serves no functional role. We establish that Cys residues in CBS1, CBS2, and the nucleotide-binding domain are more accessible for cross-linking at high than low ionic strength, indicating that these domains undergo conformational changes when transiting between the active and inactive state. Structural analyses suggest that the cystathionine beta-synthase module is largely unstructured. Moreover, we could substitute CBS1 by a linker and preserve ionic regulation of transport. These data suggest that CBS1 serves as a linker and the structured CBS2-CBS2 interface forms a hinge point for ionic strength-dependent rearrangements that are transmitted to the nucleotide-binding domain and thereby affect translocation activity.

Lactococcus lactis , a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3′,3′-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (Δψ). Wild-type cells, grown aerobically in the presence of heme, generated a Δψ even in the presence of the F 1 -F o ATPase inhibitor N , N ′-dicyclohexylcarbodiimide, while a cytochrome bd -negative mutant strain (CydAΔ) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydAΔ cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the L. lactis ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.

The ATPase subunit of the osmoregulatory ATP- binding cassette transporterOpuAfrom Lactococcus lactis has a C- terminal extension, the tandem cystathionine beta- synthase ( CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress ( Biemans- Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. ( 2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624 - 10629). C- terminal of the tandem CBS domain is an 18- residue anionic tail ( DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside- out reconstituted wild- type OpuA and the anionic tail deletion mutant OpuA Delta 12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was similar to 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg2+ and Ba2+) were more effective in activating OpuA than NH4+, K+, Na+, or Li+, consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb+ and Cs+ were potent inhibitors of wild- type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb+ and Cs+ were no longer inhibitory in OpuA Delta 12, indicating that the anionic C- terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuA Delta 12, wild- type OpuA required substantially less potassium ions ( the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C- terminal anionic tail.