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.

Crystal structure of a substrate-free aspartate transporter

“…Structural information is available for the glutamate transporter homolog Glt Ph from the archaeon Pyrococcus horikoshii, which is selective for aspartate rather than glutamate and which couples the uptake of substrate to the symport of three sodium ions [3][4][5][6][7][8] . The protein is a homotrimer, with each protomer consisting of two domains.…”

“…Different crystal structures of substrate-bound Glt Ph have revealed that the trimerization domains form a stable scaffold 6,9 . The transport domains have been captured in three states: outward facing, intermediate and inward facing [5][6][7] . The structures suggest that the transport domains behave as rigid bodies and move across the membrane like an elevator to shuttle the substrate and coupled ions from one side to the other.…”

“…3 Fig. 4a) 4,5 . The two helical hairpins (HP1 and HP2) in the transport domain, which may form intra-and extracellular gates, respectively, occlude the substrate-binding site in both Glt Ph and Glt Tk (Supplementary Fig.…”

Asymmetric perturbations of signalling oligomers

“…This is well exemplified by a recent XRC analysis of a functional double mutant of Glt Ph , a glutamate transporter from Pyrococcus horikoshii (Verdon and Boudker, 2012). While previous structure determinations captured Glt Ph (Yernool et al, 2004;Boudker et al 2007) and its analogue (Reyes et al, 2009) as symmetric trimers with all their protomers facing either outward or inward, the structure of the Glt Ph -V198CA380C Hg double mutant is an asymmetric trimer with two protomers in IFS and one in an intermediate outward-facing state (iOFS) (Fig. 4A).…”

Gate Movements in Glutamate Transporters

“…Despite the above-described data indicating that Tl ϩ can interact functionally with the transporter, this cation could not support aspartate transport (2). Therefore, the assumption that Tl ϩ faithfully mimics the action of sodium (2) must be treated with caution.…”