Abstract
Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 transporters is associated with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures reveal architectural features of the human transporters, such as intra- and extracellular domains that have potential roles in transport function, regulation by lipids and post-translational modifications. The coordination of the allosteric inhibitor in the structures and the change in the transporter dynamics measured by hydrogen–deuterium exchange mass spectrometry reveal a mechanism of inhibition, in which the transporter is locked in the outward-facing states of the transport cycle. Our results provide insights into the molecular mechanisms underlying the function and pharmacology of human SLC1 transporters.
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Acknowledgements
We thank O. Boudker for comments on the manuscript and discussion on consensus mutagenesis; P. V. Krasteva for comments on the manuscript; A. Haouz and the staff at the crystallogenesis core facility of the Institut Pasteur for assistance with crystallization screens; Staff at Synchrotron SOLEIL and the European Synchrotron Radiation Facility for assistance with data collection; D. O’Brien for discussion of HDX results. The work was funded by the ERC Starting grant 309657 (N.R.). Further support from G5 Institut Pasteur funds (N.R.), CACSICE grant (ANR-11-EQPX-008), and CNRS UMR3528 (N.R., J.C.-R.) is acknowledged.
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J.C.C.-T. and R.A. optimized and performed protein expression, purification and crystallization, and R.A. performed molecular biology; J.C.C.-T., R.A. and N.R. collected crystallographic data, and J.C.C.-T., P.L. and N.R. analysed diffraction data and structures; E.C. and R.A performed and analysed uptake experiments; E.C. prepared protein samples for HDX-MS; S.B. collected and analysed HDX-MS data with help from E.C.; All authors contributed to the experimental design of the project and manuscript preparation. N.R. conceived and supervised the project.
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Extended data figures and tables
Extended Data Figure 1 Alignment of human SLC1 transporters.
Amino acid sequences of EAAT1–EAAT5, ASCT1–ASCT2 and EAAT1cryst are compared. The boundaries of the α-helices (cylinders) in the TranD (orange) and ScaD (teal) seen in the EAAT1cryst structure are shown. To confer crystallizability, the region between TM3 and TM4c (arrows) from ASCT2 was transferred to a thermally stabilized EAAT1. To improve crystal formation in the absence of UPCH101, Met231Ile and Phe235Ile mutations (circles) were introduced to generate EAAT1cryst-II. These substitutions are found in EAAT2. Other residues involved in UPCH101 coordination are more conserved (triangles). Sequences were aligned with Jalview59.
Extended Data Figure 2 EAAT1cryst and EAAT1 glutamate uptake.
a, Initial rates of l-glutamate uptake from purified EAAT1cryst reconstituted in liposomes. The solid line is the fit of a Michaelis–Menten equation to the data with Km = 21 ± 10 μM and Vmax = 13 ± 1 pmol μg−1 protein min−1. The graph is the mean of three independent experiments, and error bars represent s.e.m. b, l-glutamate uptake was measured in HEK293F cells expressing wild-type EAAT1 (black circles) and a truncated mutant beyond Glu501 (red symbols). The initial rate of uptake decreased by approximately 2-fold in the EAAT1-truncated mutant. Data were normalized to the asymptotic level of glutamate uptake based on a monoexponential function. The rates obtained from the fits were 0.16 ± 0.03 min−1 and 0.08 ± 0.03 min−1 for EAAT1 and the truncated mutant, respectively. The graphs are means of four independent experiments performed in duplicate. Error bars represent the s.e.m.
Extended Data Figure 3 EAAT1cryst and GltPh structural comparison.
a, b, EAAT1cryst aligns to a monomer of GltPh (PDB code 2NWL), with a Cα root mean squared deviation (r.m.s.d.) value of 1.4 Å. The ScaDs (EAAT1cryst teal, and GltPh purple; a), and TranDs (EAAT1cryst orange, and GltPh purple; b) are shown separately for clarity of display.
Extended Data Figure 4 EAAT1cryst trimeric interface.
a, b, Interface of three ScaDs of the EAAT1cryst UCPH101-bound structure viewed from the extracellular side (a) and from the membrane (b). The TranDs are not shown. The ScaD of one monomer (black) buries 3,000 Å2 in the trimerization interface through extensive contacts with the two other subunits (teal and purple surfaces), including six intermolecular salt bridges (shown as green sticks for the monomer in black). The surface area buried at the trimeric interface in the other two monomers is coloured light pink. Only residues that contribute ≥10 Å2 of buried surface area are highlighted.
Extended Data Figure 5 TranD–ScaD interface.
a, b, EAAT1cryst monomer viewed from the membrane (solid black line). Residues in the TranD (coloured black) bury 1,760 Å2 at the interface with the ScaD (a). This interface extends to the extracellular side of the transporter through interactions between HP2–TM4 (sticks and pseudo-transparent spheres) (b). c, Cytoplasmic view of the monomer displaying the salt bridge between TM7 and TM5.
Extended Data Figure 6 Superposition of EAAT1cryst and EAAT1cryst-II structures.
a, b, The transport domains of EAAT1cryst (teal) and EAAT1cryst-II (pink) UCPH101-bound structures superimpose accurately after aligning their scaffold domains (a). The overall Cα r.m.s.d. value was 0.3 Å. However, the same alignment done with EAAT1cryst-II UCPH101-bound and -unbound structures shows a small but global movement of the transport domain (b), with a small increase in the overall Cα r.m.s.d. of 0.1 Å. c, d, Anomalous difference Fourier maps contoured at 2.8σ (pink mesh), from data collected at low energy X-rays (1.77 Å), show the correct sequence registry in both the TranD (orange, a) and the ScaD (teal, b).
Extended Data Figure 7 Peptide coverage map of EAAT1cryst.
A total of 111 peptides covering 76.3% of the EAAT1cryst sequence were identified by data-independent MS/MS acquisition after 2 min digestion with immobilized pepsin. Each bar below the EAAT1cryst sequence corresponds to a unique peptide. The 57 peptides coloured blue were further selected for HDX-MS data extraction and analysis. The two additional N-terminal residues (that is, Gly and Pro) that remain after protein purification are also shown. The transmembrane helices of the TranD (orange) and the ScaD (cyan) are indicated above the sequence.
Extended Data Figure 8 UCPH101 effect on the local hydrogen exchange behaviour of EAAT1cryst.
a, HDX profiles of EAAT1cryst (see Methods) in the apo unbound (top) and UCPH101-bound (bottom) state. The relative fractional uptake determined for each peptide and at each time point is plotted as a function of peptide position. The black to red lines correspond to data acquired from 10 s up to 1 h, respectively. b, The fractional uptake difference plot was generated by subtracting the deuterium uptake values in the UCPH101-unbound from those in the bound state. Negative uptake difference indicates an UCPH101-induced decrease in amide hydrogen exchange. Each dot corresponds to an average of three independent HDX-MS experiments. The four regions (labelled 1–4) that show a statistically significant modification (Wald test; P < 0.01) of deuterium uptake upon binding of UCPH101 are highlighted in grey.
Extended Data Figure 9 HDX-MS results mapped on the crystal structure of ScaD and TranD of EAAT1cryst in the unbound and UCPH101-bound state.
The colour code at the bottom shows the average relative fractional uptake measured in both domains after 10 s (top), 10 min (middle) and 1 h (bottom) labelling. Missing regions in the crystal structure are represented by dashed lines. Peptides that show a statistically significant (Wald test; P < 0.01) modification of deuterium uptake upon UCPH101 binding are labelled. Uncovered regions are coloured light blue.
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Canul-Tec, J., Assal, R., Cirri, E. et al. Structure and allosteric inhibition of excitatory amino acid transporter 1. Nature 544, 446–451 (2017). https://doi.org/10.1038/nature22064
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