Abstract
Glutamate transporters are integral membrane proteins that catalyse a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, respectively. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homologue from Pyrococcus horikoshii, GltPh, which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of GltPh in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which GltPh and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.
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Acknowledgements
We thank D. Patel and H. Li for the help with isothermal titration calorimetry and H. Weinstein for constructive criticism. X-ray diffraction data were measured at X25 beamline at the National Synchrotron Light Source. This work was supported by the National Institute of Health (O.B.) and by a Jane Coffin Childs Memorial Fund postdoctoral fellowship (N.R.).
Author Contributions N.R. and O.B. designed the project. N.R. performed protein purification, crosslinking, crystallization and structure determination. C.G. performed cloning, cell culture and protein purification. N.R. and O.B. wrote the manuscript.
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This file contains Supplementary Figures 1- 4 with Legends and Supplementary Table 1. (PDF 2674 kb)
Supplementary Movie 1
In this movie file a morph between the WT and GltPh-55C/364CHg structures is shown for a single protomer in cartoon representation. TM1 is colored blue and may serve as a reference for the approximate position of the lipid bilayer with the extracellular space on the top and the cytoplasm at the bottom. HP1 and HP2 are emphasized and colored yellow and red, respectively. Residues 55 and 364, which are cross-linked in GltPh-55C/364CHg, are shown as spheres and colored grey. Substrate L-asp, bound Na+ ions (green) and highly conserved glycines in TM2-and TM5-6 loops are also shown as spheres. (MOV 13297 kb)
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Reyes, N., Ginter, C. & Boudker, O. Transport mechanism of a bacterial homologue of glutamate transporters . Nature 462, 880–885 (2009). https://doi.org/10.1038/nature08616
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DOI: https://doi.org/10.1038/nature08616
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