Coupled ion binding and structural transitions along the transport cycle of glutamate transporters

Abstract

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.DOI: http://dx.doi.org/10.7554/eLife.02283.001

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

“…Distinct local conformational changes forming the binding pocket for aspartate were proposed by molecular dynamic simulations (17,18) and binding studies on Glt Ph (27,28). Comparison of the recent crystal structures of apoGlt Ph (29) and the closely related protein apoGlt Tk (6) with the Na ϩ -and aspartatebound proteins (8,9) are also consistent with a large local conformational change associated with sodium binding. Because the tryptophan residue in F273W is located in close proximity to the Na ϩ binding sites, this possibility is plausible.…”

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

“…Reduced dynamics of the substrate-bound transport domain has also been observed in sm-FRET studies (8,16). At present, we do not know why the dynamics are reduced, but we note that substrate binding is associated with minor conformational changes (10) and changes in the overall charge of the transport domain. Together, these values yield an energy landscape describing elevator domain activation and transmembrane motion (Fig.…”

“…The transporter forms a bowl-shaped homotrimer, in which each protomer constitutes a rigid central trimerization domain [transmembrane segments (TMs) 1, 2, 4, 5] and a peripheral transport domain (TMs 3, 6, 7, and 8, and helical hairpins 1 and 2). Aspartate (Asp) is cotransported with three sodium (Na + ) ions across the cell membrane upon a global conformational change between outward-and inward-facing states (6,(8)(9)(10)(11)(12). In contrast to other transporters that work by the rocker-switch (13) or the gated-pore (14) mechanisms, Glt Ph mediates transport by an elevator (6) mechanism in which the transport domain travels nearly 2 nm across the membrane (reviewed in ref.…”

Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters

“…Apparently, the smaller side chain of Thr at position 362 of HP2 also accommodates the occlusion of glutamate, and yet the mutant transporter cannot mediate one or more of the subsequent steps of the transport cycle such as the translocation by the so-called elevator movement (15,25). In particular, the relative positioning or internal structure of HP2 is likely to be sensitive to the bound substrate, as illustrated by a comparison of inward-facing apo and inward-facing Asp-bound structures of Glt Ph (10,14), where the position of Met-362 changes by ϳ3 Å. Moreover, Met-362 forms contacts with TM2 in these inward-facing states, specifically with Ile-61 and Val-62, which are replaced by the relatively similar residues Leu and Ile, respectively, in EAAT1-4.…”