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Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter

Nature volume 445, pages 387–393 (2007)Cite this article

Abstract

Secondary transporters are integral membrane proteins that catalyse the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of GltPh, a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-β-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound α-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.

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Figure 1: Glt Ph is an aspartate-specific sodium-driven transporter.
Figure 2: HP2 is the extracellular gate.
Figure 3: Binding of ligands and sodium to GltPh.
Figure 4: Sodium-binding sites in GltPh.
Figure 5: Sodium-binding motif.
Figure 6: Schematic mechanism illustrated by a diagram of a single subunit.

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Acknowledgements

We thank J. Mindell for support, and B. Hille and R. MacKinnon for constructive criticism. X-ray diffraction data were measured at beamlines X4A and X29 at the National Synchrotron Light Source and 8.2.2 at the Advanced Light Source. This work was supported by a National Research Service Award postdoctoral fellowship (D.Y.) and by the National Institutes of Health (E.G). E.G. is an Investigator with the Howard Hughes Medical Institute.

The coordinates for the lithium-bound native (NAT), TBOA-bound (TB) and sodium-bound (NA) states are deposited in the Protein Data Bank under accession codes 2NWL, 2NWW and 2NWX, respectively.

Author information

Author notes

  1. Olga Boudker

    Present address: Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York, 10021, USA

  2. Renae M. Ryan

    Present address: National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, Maryland, 20892, USA

  3. Dinesh Yernool

    Present address: Department of Biological Sciences, Purdue University, West Lafayette, Indiana, 47907, USA

  4. Eric Gouaux

    Present address: Vollum Institute and Howard Hughes Medical Institute, Oregon Health and Science University, Portland, Oregon, 97239, USA

  5. Olga Boudker and Renae M. Ryan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York, 10032, USA

    Olga Boudker, Renae M. Ryan, Dinesh Yernool & Eric Gouaux

  2. Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, New York, 10032, USA

    Eric Gouaux

  3. Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Misima-gun, Osaka, 618-8503, Japan

    Keiko Shimamoto

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Correspondence to Eric Gouaux.

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Boudker, O., Ryan, R., Yernool, D. et al. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445, 387–393 (2007). https://doi.org/10.1038/nature05455

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