Structural basis for substrate specificity of an amino acid ABC transporter

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate a variety of substrates, ranging from ions to macromolecules, either out of or into the cytosol (hence defined as importers or exporters, respectively). It has been demonstrated that ABC exporters and importers function through a common mechanism involving conformational switches between inward-facing and outward-facing states; however, the mechanism underlying their functions, particularly substrate recognition, remains elusive. Here we report the structures of an amino acid ABC importer Art(QN)2 from Thermoanaerobacter tengcongensis composed of homodimers each of the transmembrane domain ArtQ and the nucleotide-binding domain ArtN, either in its apo form or in complex with substrates (Arg, His) and/or ATPs. The structures reveal that the straddling of the TMDs around the twofold axis forms a substrate translocation pathway across the membrane. Interestingly, each TMD has a negatively charged pocket that together create a negatively charged internal tunnel allowing amino acids carrying positively charged groups to pass through. Our structural and functional studies provide a better understanding of how ABC transporters select and translocate their substrates.

Purification, crystallization and preliminary crystallographic analysis of a ribosome-recycling factor fromThermoanaerobacter tengcongensis(TteRRF)

Ribosome-recycling factor (RRF) plays an essential role in the fourth step of protein synthesis in prokaryotes. RRF combined with elongation factor G (EF-G) disassembles the post-termination ribosome complex and recycles the protein synthesis machine for the next round of translation. A reductive-methylation-modified RRF fromThermoanaerobacter tengcongensis(TteRRF) has been crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.1 Mcitric acid pH 3.5, 3.0 MNaCl and 50 mg ml−1methylated protein solution at 289 K. A complete data set was collected from a crystal to 2.80 Å resolution using synchrotron radiation at 100 K. The crystal belonged to space groupP6122/P6522 with unit-cell parametersa=b= 103.26,c= 89.17 Å. The asymmetric unit was estimated to contain one molecule ofTteRRF.