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.
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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).
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.
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
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.
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.
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).
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).
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.
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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