A new subfamily of bacterial glutamate/aspartate receptors (original) (raw)
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Coupled ion binding and structural transitions along the transport cycle of glutamate transporters
Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward-and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues.
Receptor–transporter interactions of canonical ATP-binding cassette import systems in prokaryotes
European Journal of Cell Biology, 2012
ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium.
PLoS ONE, 2012
Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na + ions, one H + and the counter-transport of one K + ion. Transport by an archaeal homologue of the human glutamate transporters, Glt Ph , whose three dimensional structure is known is also coupled to three Na + ions but only two Na + ion binding sites have been observed in the crystal structure of Glt Ph . In order to fully utilize the Glt Ph structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt Ph and accurately determine the number and location of Na + ions coupled to transport. Several sites have been proposed for the binding of a third Na + ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt Ph and reveal a new site for the third Na + ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt Ph , and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na + compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na + ion in Glt Ph and EAAT1.
Journal of Biological Chemistry, 2003
ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Delé age, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539 -544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry, 41, 13989 -14000). The equivalent glutamate residue (Glu 504 ) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC)
European Journal of Biochemistry, 1988
We have studied the mechanism of L-glutamate/L-aspartate transport in a fermentative oral bacterium of Streptococcus mutans (strain Ingbritt). The transport rate stays virtually constant throughout the pH range 5.5 -8.5 and followed Michaelis-Menten type kinetics. At high pH values from 7 to 8.5, transport was essentially insensitive to N,M-dicyclohexyl-carbodiimide (DCCD), an inhibitor of ATPase, and to carbonyl cyanide-ptrifluoromethoxyphenyl-hydrazone (FCCP), an ionophore dissipating proton motive force indicating that S. mutans transports glutamate by a primary transport system at the expense of ATP or an alternative energized metabolite. At lower external pH (7 -5 . 9 , DCCD (100 pM) or FCCP (10 pM) significantly inhibited L-glutamate transport while the intracellular ATP level was hardly affected, indicating that the activity of the primary transport sytem was decreased at lower intracellular pH. The glutamate transport was stimulated in the presence of potassium ion at an external pH of 6. The stimulation can be explained partly by the regulation of intracellular pH with concomitant potassium ion movement.