Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis

Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh. In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2. Our data, combined with previous studies, provide the molecular sequence of events in the coupled binding of sodium and aspartate to GltPh.