title: Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter.
abstract: Secondary transporters are integral membrane proteins that catalyse the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of Glt(Ph), a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-beta-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound alpha-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Boudker
authors.forename: Renae M
authors.initials: RM
authors.lastname: Ryan
authors.forename: Dinesh
authors.initials: D
authors.lastname: Yernool
authors.forename: Keiko
authors.initials: K
authors.lastname: Shimamoto
authors.forename: Eric
authors.initials: E
authors.lastname: Gouaux
mesh_terms: D020816:Amino Acid Motifs; D026905:Amino Acid Transport Systems / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D043902:Pyrococcus horikoshii / Q000737:chemistry*; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D026905:Amino Acid Transport Systems; D008024:Ligands; D021122:Protein Subunits; C120673:benzyloxyaspartate; D001224:Aspartic Acid; D012964:Sodium