Article | Published:

Transport domain unlocking sets the uptake rate of an aspartate transporter

Nature volume 518, pages 6873 (05 February 2015) | Download Citation

Abstract

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these ‘elevator-like’ transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the ‘humanizing’ mutations favour structurally ‘unlocked’ intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

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Accessions

Primary accessions

Protein Data Bank

Data deposits

The crystallographic model has been deposited in the Protein Data Bank under accession number 4X2S.

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Acknowledgements

The authors would like to thank P. Borbat for help with DEER data collection; H. Zhao for his help with fluorophore synthesis; G. Verdon, G. G. Gregario and S. Oh for discussions. The authors acknowledge the staff of X29 beamline at National Synchrotron Light Source and the computational resources at the Texas Advanced Computing Center at the University of Texas at Austin and the National Energy Research Scientific Computing Center, supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. The work was supported in part by the National Institute of Health grants 5U54GM087519 (to O.B. and H.W.); P01DA012408 (to H.W.); R01GM098859 and R21MH099491 (to S.C.B.); P41GM103521 and R010EB003150 (to J.H.F.).

Author information

Affiliations

  1. Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10065, USA

    • Nurunisa Akyuz
    • , Zhou Zhou
    • , Sebastian Stolzenberg
    • , Michel A. Cuendet
    • , George Khelashvili
    • , Roger B. Altman
    • , Daniel S. Terry
    • , Harel Weinstein
    • , Olga Boudker
    •  & Scott C. Blanchard
  2. National Biomedical Center for Advanced ESR Technology, Cornell University, Ithaca, New York 14853, USA

    • Elka R. Georgieva
    •  & Jack H. Freed
  3. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA

    • Elka R. Georgieva
    •  & Jack H. Freed
  4. Swiss Institute of Bioinformatics, Quartier Sorge - Batiment Genopode, 1015 Lausanne, Switzerland

    • Michel A. Cuendet
  5. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, 1305 York Avenue, New York, New York 10065, USA

    • Harel Weinstein
  6. Tri-Institutional Training Program in Chemical Biology, 445 East 69th Street, New York, New York 10065, USA

    • Scott C. Blanchard

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Contributions

N.A., O.B. and S.C.B. designed the study. N.A. designed and performed the majority of the experiments and simulated smFRET data. N.A. analysed the smFRET data, with support from D.S.T. and S.C.B. N.A. and O.B. analysed crystallographic data. E.R.G. performed and analysed DEER experiments and E.R.G. and J.H.F. interpreted the data. Z.Z. synthesized the 4S(COT)-maleimide cyanine dyes. D.S.T. made improvements to the smFRET instrumentation and analysis software. R.B.A. prepared reagents for smFRET experiments. H.W. and M.A.C. designed, and S.S., G.K., and M.A.C. carried out the molecular dynamics simulations. N.A., O.B. and S.C.B., H.W. and M.A.C. interpreted results and wrote the manuscript.

Competing interests

The authors S.C.B. and R.B.A. have equity interest in Lumidyne Technologies.

Corresponding authors

Correspondence to Olga Boudker or Scott C. Blanchard.

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DOI

https://doi.org/10.1038/nature14158

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