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Conformational heterogeneity of the aspartate transporter GltPh

Nature Structural & Molecular Biology volume 20, pages 210–214 (2013)Cite this article

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Abstract

GltPh is a Pyrococcus horikoshii homotrimeric Na+-coupled aspartate transporter that belongs to the glutamate transporter family. Each protomer consists of a trimerization domain involved in subunit interaction and a transporting domain with the substrate-binding site. Here, we have studied the conformational changes underlying transport by GltPh using EPR spectroscopy. The trimerization domains form a rigid scaffold, whereas the transporting domains sample multiple conformations, consistent with large-scale movements during the transport cycle. Binding of substrates changed the occupancies of the different conformational states, but the domains remained heterogeneous. The membrane environment favored conformations different from those observed in detergent micelles, but the transporting domain remained structurally heterogeneous in both environments. We conclude that the transporting domains sample multiple conformational states with substantial occupancy regardless of the presence of substrate and coupling ions, consistent with equilibrium constants close to unity between the observed transporter conformations.

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Figure 1: Residues selected for EPR measurements.
Figure 2: Interprotomer distances in the trimerization and the transporting domains in detergent solution.
Figure 3: Intra-protomer distances between the trimerization domain and the transporting domain.
Figure 4: Interprotomer distances in the trimerization and transporting domains in proteoliposomes.

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Acknowledgements

We thank R.H. Duurkens for performing uptake experiments, C. Rickert, D. Klose and J. Klare for help with the EPR measurements and B. Poolman for constructive criticism. This work was supported by a research fellowship and by a research grant from the Deutsche Forschungsgemeinschaft (HA 6322/1-1 to I.H. and STE 640/10, SFB944 to D.W. and H.-J.S.), the Netherlands Organisation for Scientific Research (NWO Vidi and Vici grant to D.J.S.) and the European Union (EDICT program and European Research Council starting grant to D.J.S.).

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Authors and Affiliations

  1. Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Groningen, The Netherlands

    Inga Hänelt & Dirk Jan Slotboom

  2. Department of Physics, University of Osnabrück, Osnabrück, Germany

    Dorith Wunnicke & Heinz-Jürgen Steinhoff

  3. Swiss Federal Institute of Technology Zurich, Laboratory of Physical Chemistry, Zurich, Switzerland

    Enrica Bordignon

  4. Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands

    Dirk Jan Slotboom

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  1. Inga Hänelt
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  2. Dorith Wunnicke
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  4. Heinz-Jürgen Steinhoff
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  5. Dirk Jan Slotboom
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Contributions

I.H. and D.J.S. designed the experiments. I.H., D.W. and E.B. conducted the experiments. All authors contributed to writing the manuscript and analyzing the data.

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Correspondence to Dirk Jan Slotboom.

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Hänelt, I., Wunnicke, D., Bordignon, E. et al. Conformational heterogeneity of the aspartate transporter GltPh. Nat Struct Mol Biol 20, 210–214 (2013). https://doi.org/10.1038/nsmb.2471

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