Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural insights into the inhibition of glycine reuptake

Nature volume 591, pages 677–681 (2021)Cite this article

Subjects

Abstract

The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1,2,3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Stabilization, binding and recognition of inhibitor Cmpd1 by human GlyT1.
Fig. 2: Inhibition of glycine uptake and binding mode of Cmpd1 at inward-open GlyT1.
Fig. 3: Binding pocket.
Fig. 4: Mechanism of inhibition of GlyT1.

Similar content being viewed by others

Data availability

Coordinates and structure factors for the structures of GlyT1 at 3.4 Å and 3.9 Å resolution have been deposited in the Protein Data Bank (https://www.rcsb.org) under accession codes 6ZBV and 6ZPL, respectively. The executable Ctrl-d is available via webapps.embl-hamburg.de.

References

  1. Harvey, R. J. & Yee, B. K. Glycine transporters as novel therapeutic targets in schizophrenia, alcohol dependence and pain. Nat. Rev. Drug Discov. 12, 866–885 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Grenningloh, G. et al. The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature 328, 215–220 (1987).

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Johnson, J. W. & Ascher, P. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325, 529–531 (1987).

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Cioffi, C. L. Glycine transporter-1 inhibitors: a patent review (2011–2016). Expert Opin. Ther. Pat. 28, 197–210 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Kristensen, A. S. et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol. Rev. 63, 585–640 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Gomeza, J. et al. Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition. Neuron 40, 785–796 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Cubelos, B., Giménez, C. & Zafra, F. Localization of the GLYT1 glycine transporter at glutamatergic synapses in the rat brain. Cereb. Cortex 15, 448–459 (2005).

    Article  PubMed  Google Scholar 

  8. Cubelos, B., González-González, I. M., Giménez, C. & Zafra, F. The scaffolding protein PSD-95 interacts with the glycine transporter GLYT1 and impairs its internalization. J. Neurochem. 95, 1047–1058 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Kantrowitz, J. T. & Javitt, D. C. N-methyl-d-aspartate (NMDA) receptor dysfunction or dysregulation: the final common pathway on the road to schizophrenia? Brain Res. Bull. 83, 108–121 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pinard, E., Borroni, E., Koerner, A., Umbricht, D. & Alberati, D. Glycine transporter type I (GlyT1) inhibitor, bitopertin: a journey from lab to patient. CHIMIA Int. J. Chem. 72, 477–484 (2018).

    Article  CAS  Google Scholar 

  11. Shim, S. S., Hammonds, M. D. & Kee, B. S. Potentiation of the NMDA receptor in the treatment of schizophrenia: focused on the glycine site. Eur. Arch. Psychiatry Clin. Neurosci. 258, 16–27 (2007).

    Article  PubMed  Google Scholar 

  12. Pinard, E. et al. Selective GlyT1 inhibitors: discovery of [4-(3-fluoro-5-trifluoromethylpyridin-2-yl)piperazin-1-yl][5-methanesulfonyl-2-((S)-2,2,2-trifluoro-1-methylethoxy)phenyl]methanone (RG1678), a promising novel medicine to treat schizophrenia. J. Med. Chem. 53, 4603–4614 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Krystal, J. H. et al. Neuroplasticity as a target for the pharmacotherapy of anxiety disorders, mood disorders, and schizophrenia. Drug Discov. Today 14, 690–697 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. D’Souza, D. C. et al. Dose-related target occupancy and effects on circuitry, behavior, and neuroplasticity of the glycine transporter-1 inhibitor PF-03463275 in healthy and schizophrenia subjects. Biol. Psychiatry 84, 413–421 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Jardetzky, O. Simple allosteric model for membrane pumps. Nature 211, 969–970 (1966).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Kazmier, K. et al. Conformational dynamics of ligand-dependent alternating access in LeuT. Nat. Struct. Mol. Biol. 21, 472–479 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Malinauskaite, L. et al. A mechanism for intracellular release of Na+ by neurotransmitter/sodium symporters. Nat. Struct. Mol. Biol. 21, 1006–1012 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Penmatsa, A., Wang, K. H. & Gouaux, E. X-ray structure of dopamine transporter elucidates antidepressant mechanism. Nature 503, 85–90 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Coleman, J. A. et al. Serotonin transporter–ibogaine complexes illuminate mechanisms of inhibition and transport. Nature 569, 141–145 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gotfryd, K. et al. X-ray structure of LeuT in an inward-facing occluded conformation reveals mechanism of substrate release. Nat. Commun. 11, 1005 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Singh, S. K., Yamashita, A. & Gouaux, E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448, 952–956 (2007).

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Coleman, J. A., Green, E. M. & Gouaux, E. X-ray structures and mechanism of the human serotonin transporter. Nature 532, 334–339 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Malinauskaite, L. et al. A conserved leucine occupies the empty substrate site of LeuT in the Na+-free return state. Nat. Commun. 7, 11673 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Alberati, D. et al. Glycine reuptake inhibitor RG1678: a pharmacologic characterization of an investigational agent for the treatment of schizophrenia. Neuropharmacology 62, 1152–1161 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Pinard, E. et al. Discovery of benzoylisoindolines as a novel class of potent, selective and orally active GlyT1 inhibitors. Bioorg. Med. Chem. Lett. 20, 6960–6965 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Jolidon, S., Narquizian, R., Norcross, R. D. & Pinard, E. Heterocyclic substituted phenyl methanones as inhibitors of the glycine transporter 1. WIPO patent WO/2006/082001 (2006).

  27. Brown, A. et al. Discovery and SAR of Org 24598—a selective glycine uptake inhibitor. Bioorg. Med. Chem. Lett. 11, 2007–2009 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Zimmermann, I. et al. Synthetic single domain antibodies for the conformational trapping of membrane proteins. eLife 7, e34317 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Abramson, J. & Wright, E. M. Structure and function of Na+-symporters with inverted repeats. Curr. Opin. Struct. Biol. 19, 425–432 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. LeVine, M. V. et al. The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor. Proc. Natl Acad. Sci. USA 116, 15947–15956 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Carland, J. E. et al. Molecular determinants for substrate interactions with the glycine transporter GlyT2. ACS Chem. Neurosci. 9, 603–614 (2018).

    Article  CAS  PubMed  Google Scholar 

  32. Focht, D. et al. A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition. EMBO J. 40, e105164 (2020).

    PubMed  PubMed Central  Google Scholar 

  33. Jaeger, K. et al. Structural basis for allosteric ligand recognition in the human CC chemokine receptor 7. Cell 178, 1222–1230 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Vandenberg, R. J., Shaddick, K. & Ju, P. Molecular basis for substrate discrimination by glycine transporters. J. Biol. Chem. 282, 14447–14453 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Werdehausen, R. et al. Lidocaine metabolites inhibit glycine transporter 1: a novel mechanism for the analgesic action of systemic lidocaine? Anesthesiology 116, 147–158 (2012).

    Article  CAS  PubMed  Google Scholar 

  36. Jacobs, M. T., Zhang, Y.-W., Campbell, S. D. & Rudnick, G. Ibogaine, a noncompetitive inhibitor of serotonin transport, acts by stabilizing the cytoplasm-facing state of the transporter. J. Biol. Chem. 282, 29441–29447 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Bugarski-Kirola, D. et al. Bitopertin in negative symptoms of schizophrenia-results from the phase III FlashLyte and DayLyte studies. Biol. Psychiatry 82, 8–16 (2017).

    Article  CAS  PubMed  Google Scholar 

  38. Martin-Facklam, M. et al. Glycine transporter type 1 occupancy by bitopertin: a positron emission tomography study in healthy volunteers. Neuropsychopharmacology 38, 504–512 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Weber, F. et al. Brain shuttle antibody for Alzheimer’s disease with attenuated peripheral effector function due to an inverted binding mode. Cell Rep. 22, 149–162 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Olivares, L., Aragón, C., Giménez, C. & Zafra, F. The role of N-glycosylation in the targeting and activity of the GLYT1 glycine transporter. J. Biol. Chem. 270, 9437–9442 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Gati, C. et al. Serial crystallography on in vivo grown microcrystals using synchrotron radiation. IUCrJ. 1, 87–94 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zander, U. et al. MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines. Acta Crystallogr. D 71, 2328–2343 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Popov, A. N. & Bourenkov, G. Dozor (European Synchrotron Radiation Facility, 2016).

  44. Tange, O. GNU Parallel: the command-line power tool. The USENIX Magazine 36, 42–47 (2011).

    Google Scholar 

  45. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bricogne, G. et al. BUSTER v.2.10.3 (Global Phasing, 2019).

  48. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D 74, 519–530 (2018).

    Article  CAS  Google Scholar 

  49. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).

    Article  CAS  Google Scholar 

  50. Hattori, M., Hibbs, R. E. & Gouaux, E. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20, 1293–1299 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Alexandrov, A. I., Mileni, M., Chien, E. Y. T., Hanson, M. A. & Stevens, R. C. Microscale fluorescent thermal stability assay for membrane proteins. Structure 16, 351–359 (2008).

    Article  CAS  PubMed  Google Scholar 

  52. Hawkins, P. C. D., Skillman, A. G. & Nicholls, A. Comparison of shape-matching and docking as virtual screening tools. J. Med. Chem. 50, 74–82 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Molecular Operating Environment (MOE) 2019.01 (Chemical Computing Group, 2019).

  54. Jones, G., Willett, P., Glen, R. C., Leach, A. R. & Taylor, R. Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, 727–748 (1997).

    Article  CAS  PubMed  Google Scholar 

  55. Mosca, R. & Schneider, T. R. RAPIDO: a web server for the alignment of protein structures in the presence of conformational changes. Nucleic Acids Res. 36, W42–W46 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Caulfield, W. L. et al. The first potent and selective inhibitors of the glycine transporter type 2. J. Med. Chem. 44, 2679–2682 (2001).

    Article  CAS  PubMed  Google Scholar 

  57. Madeira, F. et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 47 (W1), W636–W641 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44 (W1), W344–W350 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kantcheva, A. K. et al. Chloride binding site of neurotransmitter sodium symporters. Proc. Natl Acad. Sci. USA 110, 8489–8494 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  61. Zhang, Y.-W. et al. Chloride-dependent conformational changes in the GlyT1 glycine transporter. Proc. Natl Acad. Sci. USA (in the press) (2021).

  62. Singh, S. K., Piscitelli, C. L., Yamashita, A. & Gouaux, E. A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322, 1655–1661 (2008).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  63. Diederichs, K., & Karplus, P. A. Improved R-factors for diffraction data analysis in macromolecular crystallography. Nat. Struct. Mol. Biol. 4, 269–275 (1997).

    Article  CAS  Google Scholar 

  64. Diederichs, K., & Karplus, P. A. Linking crystallographic model and data quality. Science. 336, 1030–1033 (2012).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Thoma (F. Hoffmann-La Roche) for long-term project support and discussions; L. Hetemann and J. Gera for early contributions to producing the mutants; J. Thornton for advice and discussions on the project; F. Dall’Antonia for writing the Ctrl-d script; J. Pieprzyk for assistance with mammalian cell expression; H. Poulsen for assistance with electrophysiology studies; M. M. Garcia Alai for access to sample preparation and crystallization facilities at EMBL Hamburg; and I. M. Nemtanu and C. Guenther for technical assistance. We acknowledge the support offered at the P14 beamline operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg). We thank J. A. Lyons, B. Pedersen, C. Löw and the PROMEMO Center of Excellence of the Danish Research Foundation for discussions. This project has received funding from Novo Nordisk Foundation (to P.N.), the Lundbeck Foundation via the DANDRITE Neuroscience Center, and F. Hoffmann-La Roche. A.S. was supported by a fellowship from the EMBL Interdisciplinary Postdoc (EIPOD) programme under Marie Sklodowska-Curie Actions COFUND (grant agreement number 664726).

Author information

Authors and Affiliations

  1. Danish Research Institute of Translational Neuroscience—DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Azadeh Shahsavar & Poul Nissen

  2. European Molecular Biology Laboratory, Hamburg Unit c/o DESY, Hamburg, Germany

    Azadeh Shahsavar, Gleb Bourenkov & Thomas R. Schneider

  3. Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation Center, Basel, Switzerland

    Peter Stohler, Martin Siegrist, Wolfgang Guba, Emmanuel Pinard & Roger J. P. Dawson

  4. Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

    Iwan Zimmermann & Markus A. Seeger

  5. Linkster Therapeutics AG, Zurich, Switzerland

    Iwan Zimmermann & Roger J. P. Dawson

  6. Department of Forensic Medicine, Aarhus University, Aarhus, Denmark

    Steffen Sinning

Authors
  1. Azadeh Shahsavar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter Stohler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gleb Bourenkov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Iwan Zimmermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martin Siegrist
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wolfgang Guba
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Emmanuel Pinard
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Steffen Sinning
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Markus A. Seeger
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Thomas R. Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Roger J. P. Dawson
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Poul Nissen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.J.P.D. initiated and designed the project with P.N. and T.R.S. at Roche, Aarhus University and EMBL. Construct design was by R.J.P.D. and construct screening was by P.S., A.S. and R.J.P.D. Expression was carried out by P.S., M.S. and A.S. Purification was carried out by P.S. and A.S. Radioligand, thermal-shift and uptake assays were performed by E.P., P.S. and S.S. Sybody was generated by I.Z., M.A.S., P.S. and R.J.P.D. Crystallization, data collection and processing, and structure refinement were carried out by A.S. Serial data collection was established by A.S., G.B. and T.R.S. Dozor and serial data processing scripts were modified by G.B. Molecular modelling and docking were done by W.G. The manuscript was written by A.S., R.J.P.D. and P.N. with contributions from T.R.S., G.B., W.G. and S.S.

Corresponding authors

Correspondence to Thomas R. Schneider, Roger J. P. Dawson or Poul Nissen.

Ethics declarations

Competing interests

P.S., M.S., W.G. and E.P. are employees of F. Hoffmann-La Roche. I.Z., R.J.P.D. and M.A.S. are co-founders and shareholders of Linkster Therapeutics AG.

Additional information

Peer review information Nature thanks Carmen Villmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 GlyT1 activity.

a, Specific [3H]glycine uptake, after 10 min of incubation, by cells transfected with the crystallization construct of GlyT1 (GlyT1Crystal) and by untransfected cells, normalized to uptake by wild-type GlyT1 (GlyT1wt). Specific uptake was determined by subtracting nonspecific uptake (with 1 μM [3H]glycine plus 10 μM Cmpd1) from total uptake (with 1 μM [3H]glycine only) and was subjected to one-sample t-tests (two-tailed, not corrected for multiple corrections). Specific uptake by GlyT1Crystal was significantly different from zero (P = 0.0185); untransfected cells, by contrast, showed no statistically significant specific glycine-transport capacity (P = 0.3764). Data points are averages from n = 5, n = 4 and n = 3 independent experiments for GlyT1wt, GlyT1Crystal and untransfected cells, respectively, each performed with 6–11 measurements. Error bars represent standard error of the mean (****P < 0.0001; **P < 0.01; *P < 0.05). b, Time-course experiments performed as in a with variable incubation times, showing that uptake increases linearly within the first 60 min for both GlyT1wt and GlyT1Crystal, consistent with the occurrence of active transport. Data were subjected to linear regression analysis, yielding r2 = 0.99 and r2 = 0.97 for GlyT1wt and GlyT1Crystal, respectively. Shown are means ± s.e.m. of normalized data points from n = 3 independent experiments, each performed in duplicate. Error bars represent s.e.m.

Extended Data Fig. 2 Atomic model and electron density map of the human GlyT1–sybody complex with bound Cmpd1.

The overall structure of the GlyT1–sybody complex (cyan) with bound Cmpd1 (green) (top right) and magnified views of separate transmembrane helices, intracellular loops and extracellular loops (below and to the left) are shown in 2Fo – Fc electron density maps (blue) countered at 1.0 r.m.s.d.

Extended Data Fig. 3 Sequence conservation of hGlyT1.

a, b, Sequence (a) and overall structure (b) of human GlyT1, coloured on the basis of ConSurf58. c, Top, disrupted interaction between conserved residues W103 (in TM1a) and Y385 (in TM6) owing to the hinge-like motion of TM1a in the inward-open structure of hGlyT1 bound to Cmpd1; bottom, overlay of inward-facing occluded MhsT (wheat) on inward-open GlyT1. d, Top, the closed extracellular gate between D528 (TM10) and R125 (TM1). Bottom, a short nonhelical region is observed in TM10 at the partially conserved Y530AAS533 sequence that supposedly allows a local flexibility for opening and closing of the extracellular gate between TM10 and TM1. e, The close packing of the extracellular vestibule around W124 in the conserved GNVWRFPY motif. f, The strictly conserved disulfide bridge (C220–C229) on EL2. g, The C-terminal tail of the transporter forms a cap over the intracellular face, stabilized by interactions with IL1 and IL5. The interacting residues are shown. h, Similar to dDAT and hSERT, TM12 of GlyT1 kinks at S620 of the G613(X6)S(X4)P625 motif conserved in eukaryotic NSS transporter. Residue S620 at the kink of TM12 is shown.

Extended Data Fig. 4 Comparison of the inward-open structures of GlyT1 and SERT.

a, Superposition of the secondary structures of GlyT1 (cyan) and SERT (orange) using the so-called scaffold helices TM3–TM4 and TM8–TM9. The TM regions with structural differences are boxed, with magnified views shown in b–d. b, The intracellular half of TM1 and extracellular half of TM7 are, by 29° and 7°, respectively, closer to the core in GlyT1 compared with the corresponding TMs in inward-open SERT, and the intracellular half of TM5 has splayed 17° further from the core. c, Halfway across the membrane, TM3 in GlyT1 is locally 5° closer to the core than in SERT. The intracellular half of TM8 has splayed by 11° further away from the core of GlyT1 compared with SERT. d, On the extracellular side, TM9 is by 7° moved away from TM12, TM10 has shifted by 5° away from TM6 and TM12 is tilted by 5.5° towards the core of GlyT1. The 11° difference at the intracellular half of TM8 is also depicted. e, The intracellular gate to the core of GlyT1 defined by TM1a and TM5 is by 4 Å more closed than that of inward-open structure of SERT. Cα atoms of the conserved residues W103 of TM1a and V315 of TM5 were used for the measurements.

Extended Data Fig. 5 Detailed view of the GlyT1–sybody interface and protein–inhibitor interactions, electron density maps of Cmpd1, and crystal packing of GlyT1 and GlyT1–Lic.

a, Sybody Sb_GlyT1#7 binds to the extracellular segment of GlyT1 through several interactions between the long complementarity-determining region 3 (CDR3), CDR2 and CDR1 of Sb_GlyT1#7 and EL2, EL4, TM5 and TM7 of the transporter. The interface of GlyT1 and sybody was analysed using contact as a part of the CCP4 program suit59. Interacting residues of CDR1 (yellow), CDR2 (orange) and CDR3 (red) of the sybody and EL2, EL4 and the extracellular ends of TM5 and TM7 of GlyT1 (cyan) are depicted. b, Left, unbiased Fo – Fc (green) and 2Fo – Fc (blue) electron density maps of Cmpd1 before placement of the inhibitor, contoured at 3.0 r.m.s.d. and 0.8 r.m.s.d., respectively. Centre, 2Fo – Fc (blue) electron density map contoured at 1.0 r.m.s.d. after placement of the inhibitor and refinement. No residual Fo – Fc density is observed above 2.0 r.m.s.d. after refinement. Right, Fo − Fc simulated annealing composite omit map49 of Cmpd1 (a prominent 11.0 r.m.s.d. signal in an unbiased difference map) at 8.2 r.m.s.d. c, Diagram showing protein–ligand interactions calculated with MOE. Several hydrogen bonds that contribute to ligand binding are shown with dotted arrows (with backbone interactions in blue and side-chain interactions in green). The π-stacking interaction between the isoindoline scaffold of the ligand and Y116 is shown. Hydrophilic residues are in purple; blue rings indicate basic groups; red rings indicate acidic groups; and hydrophobic residues are in green. d, Crystal lattice arrangements viewed from the side and top of GlyT1 (top) and of GlyT1–Lic (bottom). In GlyT1, crystal contacts exist between adjacent sybodies. In GlyT1–Lic, sybodies form the crystal contacts on the extracellular side and adjacent lichenase fusion proteins do so on the intracellular side. Dashed boxes show the locations of crystal contacts. Unit cell dimensions a, b, c in GlyT1 and GlyT1–Lic are 65.17 Å, 58.14 Å, 122.31 Å and 116.41 Å, 69.71 Å, 149.43 Å, respectively.

Extended Data Fig. 6 Effects of single mutations in residues of the inhibitor-binding pocket of GlyT1, and selectivity of Cmpd1 against GlyT2.

a, b, FSEC (a) and SPA (b) signals measured for single-mutation constructs of GlyT1 compared with the wild-type transporter at 4 °C and 50 °C (a) or 4 °C and 30 °C (b). The absence of an SPA signal for the L120A, Y196A, G373A, W376A, L379A and T472A constructs confirms the inability of the mutant to bind the inhibitor. A weak SPA signal for the G121A construct at 4 °C, and for the M382A and I399A constructs at both 4 °C and 30 °C, was measured. A relatively higher SPA signal for the Y116A mutant can be explained as the isoindoline scaffold of the inhibitor is further supported by hydrophobic interactions with surrounding residues other than Y116. Bars represent average FSEC and SPA signals in a and b, respectively (in a, shown are individual data points from n = 3 independent experiments for W376A, n = 2 for wild-type and Y116A, and n = 1 for L120A, G121A, Y196A, G373A, L379A, M382A, I399A and T472A; in b, from n = 3 for wild-type, Y116A, and I399A, n = 2 for G121A, G373A, W376A and M382A, and n = 1 for L120A, Y196A, L379A and T472A; each in n = 3 technical replicates). Error bars represent s.e.m. c, Thermostabilizing effect of the I192A mutation (introduced into the GlyT1minimal construct, which also contains N- and C- terminal deletions of residues 1–90 and 685–706) compared with GlyT1minimal, measured by FSEC–TS analysis. Apparent Tm values for GlyT1minimal and the I192A mutant were 36.6 ± 0.5 °C and 52.5 ± 1.5 °C, respectively. Bars represent average apparent Tm values, with data points from n = 2 and n = 3 independent experiments for GlyT1minimal and I192A, respectively, shown as individual circles (±s.e.m.). d, Nonbinding I192A mutation. Left, a comparable (with the value obtained by FSEC–TS analysis) apparent Tm value of 33.5 ± 0.4 °C was measured for GlyT1minimal in SPA–TS analysis, while no signal was observed for the I192A mutant (left, n = 2 independent experiments, each with triplicate measurements; shown are means ± s.e.m.). Right, SPA signals measured at 4 °C and 30 °C. The absence of a signal for I192A confirms the inability of the mutant to bind the inhibitor. Bars represent the average SPA signal, with individual data points from n = 3 technical replicates shown. The experiment was repeated independently once with similar results. e, Position of I192A (in TM3), stabilizing a rotamer of W376 (TM6) in an edge-to-face stacking interaction with Cmpd1. f, Assay for [3H]glycine-uptake inhibition in mammalian Flp-in-CHO cells transfected with human GlyT2 cDNA, showing that Cmpd1, a selective inhibitor of GlyT1, does not inhibit uptake of glycine by GlyT2. The curve was calculated from n = 4 technical replicates (individual data points are shown; whiskers extend from minimum to maximum).

Extended Data Fig. 7 Ion- and glycine-binding sites in GlyT1.

a, Cl− (light green) and Na+ (purple) ions in the GlyT1–Lic structure are shown as spheres. Fo − Fc simulated annealing composite omit maps (green mesh) for Cl− and Na+ ions (prominent peaks at 6.8 r.m.s.d. and 6.5 r.m.s.d., respectively, in an unbiased difference map, chain A) are shown at 4.0 r.m.s.d. Cmpd1 is depicted in green and the residues that are likely to coordinate the Cl− and Na+ ions are shown as sticks and with dashed lines (chain A in the asymmetric unit). The Cl− ion is coordinated by conserved residues Y142 (TM2), Q367 (TM6), S371 (in the unwound region of TM6) and S407 (TM7), similar to the Cl− site in dDAT and SERT18,22, with a mean coordination distance of 3.0 Å, and probably also by N403 (TM7)60, but with a longer coordination distance. Mutation of residues Q367 and S407 has further been shown to affect GlyT1’s response to Cl−, highlighting the involvement of these residues in Cl− binding61.The Na+ ion in the Na2 site is within a mean coordination distance of 3.1 Å from the carbonyl oxygen of the conserved residues G115, V118 (TM1) and T472 (TM8), as observed in previous structures of NSS transporters, and the carbonyl oxygen of the Cmpd1 scaffold (measured in chain A of the asymmetric unit). The Na1 site observed in other NSS structures is occupied by the methyl sulfone substituent of the inhibitor in this structure. b, The 2Fo − Fc electron density map (blue) for helices involved in ion binding is contoured at 1.0 r.m.s.d. c, d, Superposition of tryptophan-bound MhsT (c; light orange, PDB ID 4US3; ref. 17) and glycine-bound LeuT (d; purple, PDB ID 3F4J; ref. 62) on Cmpd1-bound GlyT1 (cyan). The sulfonyl moiety of the inhibitor matches with the carboxylate of tryptophan or glycine. Glycine bound to GlyT1 probably interacts with the backbone amide of L120 and G121 from TM1 and the hydroxyl group of Y196 from TM3, similar to the stabilizing interactions of LeuT and MhsT with their respective bound ligands, glycine and tryptophan. d, Scintillation proximity competition assays using [3H]Org24598 and varying concentrations of bitopertin and glycine with GlyT1minimal, showing that bitopertin and glycine compete with [3H]Org24598 at concentrations of 1.0 × 10−5 ± 1.8 × 10−6 mM and 0.1 ± 0.003 mM (means ± s.e.m. from triplicate measurements), respectively. Although direct competition between bitopertin and glycine is not shown in the experiment, the similarities of [3H]Org24598/bitopertin are nevertheless highly suggestive that bitopertin and glycine also compete with each other for binding at GlyT1. Curves were calculated from n = 3 technical replicates (individual measurements are shown; whiskers extend from minimum to maximum).

Extended Data Fig. 8 Inhibition of GlyT1-mediated glycine transport by Cmpd1 is not competitive.

Left, uptake assays using HEK293-MSR cells transfected with GlyT1wt display Michaelis–Menten kinetics that can be inhibited by Cmpd1 in a dose-dependent manner. Centre, Eadie–Hofstee plots of uptake data verify that the inhibition of GlyT1-mediated glycine uptake by Cmpd1 is not competitive (independent experiments are normalized against Vmax). Right, kinetic parameters derived from Michaelis–Menten analysis show that Cmpd1 reduces Vmax (normalized representation) in a dose-dependent manner (P = 0.0038 and P < 0.0001 for Vmax at 240 nM and 960 nM of Cmpd1, respectively), whereas Km values are mostly not altered by increasing the inhibitor concentration, except for a single concentration (P = 0.0152 at 240 nM of Cmpd1). Km and Vmax values for increasing concentrations of Cmpd1 are 156 ± 18 μM, 137 ± 17 μM, 99 ± 22 μM and 109 ± 10 μM, and 18,564 ± 3,381 CPM, 16,524 ± 3534 CPM, 9,819 ± 2,437 CPM and 5,512 ± 1,076 CPM, respectively (means ± s.e.m.). These data exclude competitive inhibition for the inhibitor. Curves were calculated from n = 4 independent experiments, each performed with duplicate measurements. Data points are the average of independent experiments. Whiskers extend from minimum to maximum. One-way analysis of variance (ANOVA), corrected according to Dunnett’s test, was used to determine whether each mean of aggregate data from four independent experiments was significantly different from the corresponding value with no inhibitor present (****P < 0.0001; **P < 0.01; *P < 0.05).

Extended Data Fig. 9 Number of scaled mini datasets per number of frames, and statistics of scaled mini datasets for GlyT1 and GlyT1–Lic crystals.

a, c, GlyT1; b, d, GlyT1–Lic. a, b, Numbers of scaled partial datasets with a given number of frames (1–41) for GlyT1 (a) and GlyT1–Lic (b) datasets. Mini datasets containing 3–20 frames were picked automatically; in several cases, mini datasets adjacent in frame numbers were manually merged into larger datasets containing more than 20 frames. c, d, Statistics of scaled mini datasets for GlyT1 (c) and GlyT1–Lic (d). Calculated by XSCALE45, I/Sigma is the mean of the reflection intensity, I, of unique reflections divided by the standard deviation of the reflection intensity, after merging symmetry-related observations. R-meas is the redundancy-independent R-factor (for intensities)63. CC(1/2) is the percentage of correlation between intensities from random half-datasets64.

Extended Data Table 1 Data collection and refinement statistics
Full size table

Supplementary information

Supplementary Figures

This file contains Supplementary Figure 1 which shows multiple sequence alignment of NSS family members and Supplementary Figure 2 - the purification of GlyT1.

Reporting Summary

Peer Review File

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shahsavar, A., Stohler, P., Bourenkov, G. et al. Structural insights into the inhibition of glycine reuptake. Nature 591, 677–681 (2021). https://doi.org/10.1038/s41586-021-03274-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-021-03274-z

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing