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Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context

Nature Structural & Molecular Biology volume 19, pages 212–219 (2012)Cite this article

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Abstract

Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-β-D-glucopyranoside (β-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT–substrate complexes in lipid bicelles and in β-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site.

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Figure 1: Experimental flow chart and lattice contacts in bicelle-based crystal forms.
Figure 2: LeuT crystal structures derived from bicelles and β-OG are similar.
Figure 3: Mapping detergent sites in LeuT.
Figure 4: LeuT has a single high-affinity substrate site in bicelles.

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Acknowledgements

We thank P. Shaffer for crystallization and measurement of diffraction data for the LeuT–β-SeHG complex; K. Wang and A. Penmatsa for assistance with LeuT expression and purifications; R. Hibbs for suggestions on structure refinement; M. Kavanaugh and D. Claxton for comments; and L. Vaskalis for assistance with illustrations. We also thank the staff at beamlines 24-ID-E and 24-ID-C of the Advanced Photon Source, and the staff at 8.2.1 and 5.0.2 of the Advanced Light Source. This work was supported by the US National Institutes of Health. E.G. is an investigator with the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Vollum Institute, Oregon Health & Science University, Portland, Oregon, USA

    Hui Wang & Eric Gouaux

  2. Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon, USA

    Johannes Elferich

  3. Howard Hughes Medical Institute, Oregon Health & Science University, Portland, Oregon, USA

    Eric Gouaux

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  1. Hui Wang
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H.W. and E.G. designed the research; H.W., J.E. and E.G. carried out the research and analyzed the data; and H.W. and E.G. wrote the paper.

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Correspondence to Eric Gouaux.

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Wang, H., Elferich, J. & Gouaux, E. Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context. Nat Struct Mol Biol 19, 212–219 (2012). https://doi.org/10.1038/nsmb.2215

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