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Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters

Abstract

Na+/Cl--dependent transporters terminate synaptic transmission by using electrochemical gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1–5 related to 6–10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.

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Acknowledgements

We appreciate the beamtime and the assistance of the personnel at beamline 8.2.2 of the Advanced Light Source and beamlines X4A, X12B and X29 of the National Synchrotron Light Source; B. Honig and L. Forrest for help with sequence alignment; M. A. Gawinowicz for mass spectrometry and free amino acid analysis; J. Moon for help with bacterial culture and crystallization; H. Furukawa for assistance with vector construction and light scattering experiments; O. Boudker for liposome reconstitution; and D. Yernool, O. Boudker and R. Ryan for comments on the manuscript. A.Y. is on leave from the Laboratory for Structural Biochemistry, RIKEN Harima Institute at SPring-8, Japan. S.K.S. is supported by an NIH NRSA postdoctoral fellowship. The work was supported by the NIH. E.G. is an investigator with the Howard Hughes Medical Institute.

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Competing interests

The coordinates for the structure have been deposited in the Protein Data Bank under the accession code 2A65. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Correspondence to Eric Gouaux.

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Supplementary Notes

This file contains Supplementary Table S1 (data collection and phasing statistics), Supplementary Methods, Supplementary Figures S1-S5 and accompanying Supplementary Figure Legends. (PDF 2016 kb)

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Further reading

Figure 1: Amino acid sequence alignment and secondary structure of LeuTAa.
Figure 2: LeuT Aa structure.
Figure 3: Leucine binding site.
Figure 4: Sodium ion binding sites.
Figure 5: Extracellular and cytoplasmic gates.
Figure 6: Speculative transport mechanism.

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