Structure and mechanism of a glutamate-GABA antiporter

Food-borne hemorrhagic Escherichia coli, exemplified by the strains O157:H7 and O104:H4 (refs 1, 2), require elaborate acid-resistance systems (ARs)(3) to survive the extremely acidic environment such as the stomach (pH approximate to 2). AR2 expels intracellular protons through the decarboxylation of L-glutamate (Glu) in the cytoplasm and exchange of the reaction product c-aminobutyric acid (GABA) with extracellular Glu. The latter process is mediated by the Glu-GABA antiporter GadC(4,5), a representative member of theamino-acid-polyamine-organocation superfamily of membrane transporters. The functional mechanism of GadC remains largely unknown. Here we show, with the use of anin vitro proteoliposome-based assay, that GadC transports GABA/Glu only under acidic conditions, with no detectable activity at pH values higher than 6.5. We determined the crystal structure of E. coli GadC at 3.1 angstrom resolution under basic conditions. GadC, comprising 12 transmembrane segments (TMs), exists in a closed state, with its carboxy-terminal domain serving as a plug to block an otherwise inward-open conformation. Structural and biochemical analyses reveal the essential transport residues, identify the transport path and suggest a conserved transport mechanism involving the rigid-body rotation of a helical bundle for GadC and other amino acid antiporters.