Fig 2: Schematic representation of conformational changes in SLC1A5 during substrate transport.(a) In the outward-facing state (top), the Zone two from the transport domain interacts with the scaffold domain, with the Zone one partially occupied by the ECL2a. Two different poses of the ECL2a were observed (shown in red and dark gray lines): one is situated at the side of the transport domain (red line); the other is crossing over the crests of ECL4 and ECL5 (dark gray line). In the inward-facing state (bottom), the ECL2a has to be repositioned, releasing the Zone one to interact with the scaffold domain. Residues from Zone one and Zone two are highlighted (Figure 1—figure supplement 5). (b) The inherent plasticity around the TM2-3 loop, ECL3, TM3, and TM6 regions (highlighted in dash lines) allows the transitions between the outward-facing (top) and inward-facing states (bottom). The relative position of transport domain in the outward-facing state is highlighted in dash (bottom panel in b). The regions of SLC1A5 are colored based on the Figure 1a. The light gray line depicts membrane boundary.

Fig 3: The substrate, L-glutamine-bound SLC1A5 structure.(a) Conformational change for the HP2 upon binding of L-glutamine in the outward-facing state of SLC1A5. The unliganded structure is shown in gray and the L-glutamine-bound structure is in navy. EM density over the ligand is shown as a blue mesh. (b and c) Zoomed in views of the substrate binding sites of the outward-facing and inward-facing states (PDB:6GCT) of SLC1A5, respectively. (d) Interaction of L-Asp in the outward-facing state of SLC1A3 (PDB code: 5LLU). Critical side chains are labeled (stick representation) with hydrogen bonds (red dashed lines). Molecular surface in the substrate binding sites are in pink.

Fig 4: Cryo-EM structure of human SLC1A5.(a) A density map of SLC1A5 homotrimer viewed from the side of the membrane (left) and the extracellular face (right) highlighting the scaffold domain (green), transport domain (navy), ECL2 (red) and N-acetyl-D-glucosamine (NAG, in orange). The density assigned to CHS is colored in magenta. (b) Micelle/membrane and trimerization interfaces are highlighted in gray and wheat, respectively. Interactions between transport and scaffold domains are illustrated with lines. (c) A schematic representation of the domains present in human SLC1A5. The N-linked glycosylation sites are indicated by orange circle. Dashed lines represent residues disordered in the structure.