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. 2020 Feb 21;11:1005. doi: 10.1038/s41467-020-14735-w

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — The structures are shown as cross-sectional representations organized clockwise after progressive opening to the inside starting with the Na⁺/substrate free outward-facing occluded LeuT state (PDB-ID: 5JAE¹²; middle right) followed by the Na⁺-bound outward-facing open LeuT state (PDB-ID: 3TT1¹¹; bottom right), the Na⁺/l-Leu-bound outward-facing occluded LeuT state (PDB-ID: 2A65¹⁰; bottom left), the Na⁺/l-Trp-bound inward-facing occluded MhsT state with an unwound TM5 (PDB-IDs: 4US3¹³; middle left and in square brackets), the Na⁺/l-Trp-bound inward-facing occluded MhsT state with helical TM5 (PDB-IDs: 4US4¹³; upper left and in square brackets), the Na⁺/l-Phe-bound inward-facing occluded state of LeuT (this study; top middle) and the apo inward-facing open LeuT state (PDB-ID: 3TT3; top right). For clarity, only the orientation of transmembrane segments (TMs) 1, 5, and 6, and extracellular loop (ECL) 4 is shown to illustrate the most significant concerted movements related to opening and closure of the intracellular and extracellular vestibules, respectively. Sodium ions and substrate molecules are shown as purple spheres and yellow sticks, respectively. The structures support a model in which Na⁺/substrate reach the outward-facing binding sites via open extracellular vestibule, leading to its closure by the conformational rearrangements involving TM1, 5 and ECL4. The adapted inward-facing occluded conformation transits subsequently towards the inward-facing state, largely by the release of the N-terminal tail, disruption of the interactions provided by internal gate residues and changes in TM5 dynamics. Specifically, the structures illustrate how changes in TM5 dynamics involving putative cytosolic unwinding followed by a progressive outward movement towards the membrane (from PDB-ID: 2A65 over PDB-ID: 4US3 and PDB-ID: 4US4 to the present structure of LeuT_W8A) putatively lead to formation of a large cytoplasmic cavity allowing access to Na2. Furthermore, as the transport cycle progresses, TM1 swings away from TM5 and 6 to open the intracellular vestibule that permits the release of bound Na⁺/substrate (PDB-ID: 3TT3). Finally, following Na⁺/substrate diffusion, TM1 and 5 pull back, and ECL4 uncaps the extracellular vestibule, facilitating the return to the apo outward-facing open state able to bind Na⁺/substrate again.