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Conformational ensemble of the sodium-coupled aspartate transporter

Nature Structural & Molecular Biology volume 20, pages 215–221 (2013)Cite this article

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Abstract

Sodium and aspartate symporter from Pyrococcus horikoshii, GltPh, is a homolog of the mammalian glutamate transporters, homotrimeric integral membrane proteins that control neurotransmitter levels in brain synapses. These transporters function by alternating between outward-facing and inward-facing states, in which the substrate binding site is oriented toward the extracellular space and the cytoplasm, respectively. Here we used double electron-electron resonance (DEER) spectroscopy to probe the structure and the state distribution of the subunits in the trimer in distinct hydrophobic environments of detergent micelles and lipid bilayers. Our experiments reveal a conformational ensemble of protomers that sample the outward-facing and inward-facing states with nearly equal probabilities, indicative of comparable energies, and independently of each other. On average, the distributions varied only modestly in detergent and in bilayers, but in several mutants unique conformations were stabilized by the latter.

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Figure 1: Spin-labeled residues.
Figure 2: Narrow distance distributions of trimerization domain residue 55.
Figure 3: Broad distance distributions measured for residues in the transport domain.
Figure 4: Comparison of measured and expected distances.
Figure 5: The inward and outward-facing states of GltPh have close energies.

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Acknowledgements

The work was supported by US National Institute of Neurological Disorders and Stroke grants NS064357 and NS064357-02S1 and a grant from Bohmfalk Charitable Trust to O.B., and US National Center for Research Recourses grant P41-RR016292, US National Institute of General Medical Sciences grant P41GM103521 and US National Institute of Biomedical Imaging and Bioengineering grant R010EB003150 J.H.F.

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Authors and Affiliations

  1. National Biomedical Center for Advanced Electron Spin Resonance Technology, Cornell University, Ithaca, New York, USA

    Elka R Georgieva, Peter P Borbat & Jack H Freed

  2. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA

    Elka R Georgieva, Peter P Borbat & Jack H Freed

  3. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA

    Christopher Ginter & Olga Boudker

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  1. Elka R Georgieva
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  2. Peter P Borbat
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Contributions

E.R.G. and O.B. developed the mutation strategy for DEER spectroscopy, and designed the molecular biology and biochemical part of the experiments. E.R.G., P.P.B. and J.H.F. designed the DEER spectroscopy experiments. E.R.G. performed most of the DEER spectroscopy experiments, protein expression and purification, and spin labeling. C.G. carried out the mutagenesis and participated in protein expression and purification. P.P.B. performed some of the DEER measurements. E.R.G., O.B. and P.P.B. analyzed the data. O.B., E.R.G., P.P.B. and J.H.F. wrote the manuscript.

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Correspondence to Olga Boudker.

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The authors declare no competing financial interests.

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Georgieva, E., Borbat, P., Ginter, C. et al. Conformational ensemble of the sodium-coupled aspartate transporter. Nat Struct Mol Biol 20, 215–221 (2013). https://doi.org/10.1038/nsmb.2494

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