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The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Förster resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the osmoregulatory type I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using this methodology, we studied ligand-binding mechanisms, as well as ATP and glycine betaine dependences of conformational changes. Our work expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Förster-resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the type-I ABC importer OpuA fromLactococcus lactis.We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using the methodology, we studied ligand-binding mechanisms as well as ATP and glycine betaine dependences of conformational changes. Our study expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open to a closed conformation upon substrate binding, providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases, similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport. ispartof: ELIFE vol:8 ispartof: location:England status: published

Solute transport via ATP binding cassette (ABC) importers involves receptor-mediated substrate binding, which is followed by ATP-driven translocation of the substrate across the membrane. How these steps are exactly initiated and coupled, and how much ATP it takes to complete a full transport cycle, are subject of debate. Here, we reconstitute the ABC importer GlnPQ in nanodiscs and in proteoliposomes and determine substrate-(in)dependent ATP hydrolysis and transmembrane transport. We determined the conformational states of the substrate-binding domains (SBDs) by single-molecule Forster resonance energy transfer measurements. We find that the basal ATPase activity (ATP hydrolysis in the absence of substrate) is mainly caused by the docking of the closed-unliganded state of the SBDs onto the transporter domain of GlnPQ and that, unlike glutamine, arginine binds both SBDs but does not trigger their closing. Furthermore, comparison of the ATPase activity in nanodiscs with glutamine transport in proteoliposomes shows that the stoichiometry of ATP per substrate is close to two. These findings help understand the mechanism of transport and the energy coupling efficiency in ABC transporters with covalently linked SBDs, which may aid our understanding of Type I ABC importers in general.

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.