Structural basis for substrate specificity of an amino acid ABC transporter

Abstract

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 inwardfacing 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.ABC transporters | substrate selectivity | inward-facing state | two binding sites | type I importer

Lipid-Uptake Pathways and Lipid-Protein interactions in P-glycoprotein Revealed by Coarse-Grained Molecular Dynamics Simulations

“…Our findings suggest that PC and PE lipids access the internal drugbinding pocket through the portals P1 and P2, which has also been reported for drug molecules in several studies [1] [4]. It has been also suggested that a positively charged ring of residues such as Arg and Lys [63] [64], could drive substrates towards the cavity, and promote lipid-protein interactions in ABC transporters in general via electrostatic interactions. From our simulations, two binding hot spots at P1 were identified: Lys230 (H4), and Arg355 (H6).…”

Alanine Metabolism inBacillus subtilis

“…Deletion of the alanine racemase gene, alrA (dal), has previously been shown to result in auxotrophy for D-alanine (9). We sought to use this property to determine if a specific D-alanine uptake system was present or if uptake was mediated by transporters with degenerate specificity as has been suggested for some other amino acid uptake systems (27). So we characterised the growth of a strain lacking the conditionally essential alanine racemase, alrA (strain RD180) in a LB growth medium.…”

From substrate specificity to promiscuity: hybrid ABC transporters for osmoprotectants

“…Genetic, biochemical and structural analysis revealed the presence of low-affinity ligand-binding sites in the TMDs of the E. coli maltose/maltodextrin system (Shuman, 1982;Covitz et al, 1994;Khare et al, 2009;Bordignon et al, 2010;Cui et al, 2010;Chen, 2013) and of the arginine/histidine ABC transporter from the thermophile T. tengcongensis (Yu et al, 2015), both of which belong to the type-I subfamily of ABC import systems (Locher, 2016;Lewinson and Livnat-Levanon, 2017). Mutational analysis of the internal binding sites in the MalF and MalG TMDs revealed that it is essential for the functioning of the maltose/maltodextrin ABC transporter (Covitz et al, 1994;Ehrle et al, 1996;Steinke et al, 2001;Khare et al, 2009).…”