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X-ray structures of LeuT in substrate-free outward-open and apo inward-open states

Abstract

Neurotransmitter sodium symporters are integral membrane proteins that remove chemical transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances.

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Figure 1: The substrate-free, Na + -bound state is outward-open.
Figure 2: Sodium sites in the outward-open state.
Figure 3: Inward-open conformation.
Figure 4: Changes in gate, substrate and ion site interactions and coupling to helix movements.
Figure 5: Schematic of transport in LeuT.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession codes 3TT1, 3TT3 and 3TU0 for the LeuTK(Y108F)–2B12 complex, the LeuTK(TSY)–6A10 complex and the LeuTK(TS) complex with alanine, respectively.

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Acknowledgements

We thank D. Cawley for monoclonal antibody production, L. Vaskalis for help with illustrations and the staff at the Advanced Photon Source beamline 24-ID-E and at the Advanced Light Source beamline 5.0.2 for their assistance with X-ray data collection and processing. We are grateful to E. Haddadian, T. Sosnick and K. Freed for assistance in refining the backbone torsional and side-chain angles using their unpublished TOP algorithm. We thank all Gouaux laboratory members, especially C. Piscitelli and S. K. Singh, for discussions and helpful suggestions throughout the project. This work was supported by the National Institutes of Health. E.G. is an investigator with the Howard Hughes Medical Institute.

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H.K. and E.G. contributed to all aspects of the project.

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Correspondence to Eric Gouaux.

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Supplementary information

Supplementary Information

The file contains Supplementary Tables 1-2, Supplementary Figures 1-13 with legends, a Supplementary Discussion and Supplementary Movie legend. (PDF 3978 kb)

Supplementary Movie 1

The movie depicts the conformational changes associated with isomerization from the open-to-out substrate-free to occluded state, to the open-to-in states (please see Supplementary Information file for full legend). (MOV 13215 kb)

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Krishnamurthy, H., Gouaux, E. X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature 481, 469–474 (2012). https://doi.org/10.1038/nature10737

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