Structural And Functional Studies Into An ABC Transporter Complex Art I-Art（QN）₂

ATP-binding cassette（ABC） transporters are ubiquitous transmembrane proteins that translocate plenty of substrates, ranging from ions to macromolecules, either into or out of the cytosol（thus defined as importers or exporters, respectively）. Minimal ABC transporters contain two transmembrane domains（TMDs） and two nucleotide binding domains（NBDs）.Canonical ABC importers additionally require the assistance of substrate binding proteins（SBPs） to transport substrates from the extracellular to the cytoplasm. It has been demonstrated that ABC importers and exporters function through a common mechanism involving conformational switches between outward- and inward-facing states. However, the mechanism underlying their functions, in particular, substrate recognition, remains elusive.Here, we solved the structure of an amino acid ABC-importer Art（QN）₂,from the Thermoanaerobacter tengcongensis, either in its apo-form or in complex with substrates（His, Arg） and/or ATP at 2.8?, 2.8?, 2.6?,3? and 2.5? resolution, respectively.Moreover, the structure of the solute binding protein, Art I, was solved in complex with Arg at 1.48 ?.Art（QN）₂ is composed of homodimers each of the the nucleotide binding domain（NBD）, ArtN and transmembrane domain（TMD）, ArtQ.The structures indicate that the straddling of the TMDs around the two-fold axis forms a substrate translocation pathway across the membrane. Interestingly, each TMD possessesa negatively-charged pocket that together create a negatively-charged internal tunnel allowing amino acids carrying positively charged groups to pass through. Functional analyses of the transporter in proteoliposomes suggest its capability to undergo substrate-dependent conformational changes resulting in stimulated ATPase activity. Taken together, our structural and functional studies provide a better understanding of how ABC transporters select and translocate their substrates.