Arginine-agmatine Reverse Membrane Transport Proteins Adic Structure And Function

AdiC is a representative member of a large superfamily called APC transporters, standing for Amino Acids, Polyamine and OrganoCation superfamily transporters. It contains more than 250 members across species, from bacteria to human being. AdiC was identified to be the Arginine:Agmatine antiporter in E. coli and other enterobacteria. It is a key component of the Acid Resistance system. Under extremely acidic environment, enteric bacteria such as Escherichia coli rely on the amino acid antiporter AdiC to uptake environmental Arginine and expel the cytosolic Agmatine. Together, each cycle of reaction consumes one proton from the cytoplasm, therefore increasing the cytosolic pH.We have designed different point mutations mainly based on the analysis of AdiC wide type structure from E. coli. We used ITC to test the binding affinity between the protein and the substrate and used transporter assay to test the transport activity.On the basis of the biochemical results, we chose AdiC-N22A as our target to do structure research. Because this mutation not just improves the binding affinity between the protein and the substrate, but also has no obviously influence on the transport activity.We solved the X-ray structure of an E. coli AdiC variant bound to Arg at 3.0 A resolution. Arg binding induces pronounced structural rearrangement in TM6 and, to a lesser extent, TM2 and TM10, resulting in an occluded conformation. The positively charged Arg is enclosed in an acidic binding chamber, with the head groups of Arg hydrogen bonded to main chain atoms of AdiC and the aliphatic portion of Arg stacked by hydrophobic side chains of highly conserved residues.Structural and biochemical analysis reveals the essential ligand-binding residues, defines the transport route, and suggests a conserved mechanism for the antiporter activity. Structural analysis identified three potential gates, involving four aromatic residues and Glu208, which may work in concert to differentially regulate the upload and release of Arg and Agm.