Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter.
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.
Boudker
Olga
O
Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USA.
Ryan
Renae M
RM
Yernool
Dinesh
D
Shimamoto
Keiko
K
Gouaux
Eric
E
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
D026905:Amino Acid Transport Systems; D008024:Ligands; D021122:Protein Subunits; C120673:benzyloxyaspartate; D001224:Aspartic Acid; D012964:Sodium