Abstract
The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants (S)-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of (S)-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design.
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Primary accessions
Protein Data Bank
Data deposits
The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under the following accession codes: ts3 paroxetine (5I6X), ts2 paroxetine (5I6Z), ts3 (S)-citalopram (5I71), ts3 (S)-citalopram (soaked) (5I73), ts3 Br-citalopram (5I74), ts3 Br-citalopram (soaked) (5I75), and 8B6 Fab (5I66).
References
Berger, M., Gray, J. A. & Roth, B. L. The expanded biology of serotonin. Annu. Rev. Med. 60, 355–366 (2009)
Rapport, M. M., Green, A. A. & Page, I. H. Serum vasoconstrictor, serotonin; isolation and characterization. J. Biol. Chem. 176, 1243–1251 (1948)
Blackburn, K. J., French, P. C. & Merrills, R. J. 5-hydroxytryptamine uptake by rat brain in vitro . Life Sci. 6, 1653–1663 (1967)
Carlsson, A., Fuxe, K. & Ungerstedt, U. The effect of imipramine on central 5-hydroxytryptamine neurons. J. Pharm. Pharmacol. 20, 150–151 (1968)
Glowinski, J. & Axelrod, J. Inhibition of uptake of tritiated-noradrenaline in the intact rat brain by imipramine and structurally related compounds. Nature 204, 1318–1319 (1964)
Hoffman, B. J., Mezey, E. & Brownstein, M. J. Cloning of a serotonin transporter affected by antidepressants. Science 254, 579–580 (1991)
Blakely, R. D. et al. Cloning and expression of a functional serotonin transporter from rat brain. Nature 354, 66–70 (1991)
Kristensen, A. S. et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol. Rev. 63, 585–640 (2011)
Bröer, S. & Gether, U. The solute carrier 6 family of transporters. Br. J. Pharmacol. 167, 256–278 (2012)
Wennogle, L. P. & Meyerson, L. R. Serotonin modulates the dissociation of [3H]imipramine from human platelet recognition sites. Eur. J. Pharmacol. 86, 303–307 (1982)
Zhong, H. et al. An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant. Neurosci. Lett. 462, 207–212 (2009)
Hahn, M. K. & Blakely, R. D. The functional impact of SLC6 transporter genetic variation. Annu. Rev. Pharmacol. Toxicol. 47, 401–441 (2007)
Andersen, J., Kristensen, A. S., Bang-Andersen, B. & Stromgaard, K. Recent advances in the understanding of the interaction of antidepressant drugs with serotonin and norepinephrine transporters. Chem. Commun. (Camb.) 3677–3692 (2009)
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)
Wang, H. et al. Structural basis for action by diverse antidepressants on biogenic amine transporters. Nature 503, 141–145 (2013)
Yamashita, A., Singh, S. K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 437, 215–223 (2005)
Singh, S. K. & Pal, A. Biophysical approaches to the study of LeuT, a prokaryotic homolog of neurotransmitter sodium symporters. Methods Enzymol. 557, 167–198 (2015)
Kazmier, K. et al. Conformational dynamics of ligand-dependent alternating access in LeuT. Nature Struct. Mol. Biol. 21, 472–479 (2014)
Wang, K. H., Penmatsa, A. & Gouaux, E. Neurotransmitter and psychostimulant recognition by the dopamine transporter. Nature 521, 322–327 (2015)
Penmatsa, A., Wang, K. H. & Gouaux, E. X-ray structures of Drosophila dopamine transporter in complex with nisoxetine and reboxetine. Nature Struct. Mol. Biol. 22, 506–508 (2015)
Penmatsa, A., Wang, K. H. & Gouaux, E. X-ray structure of dopamine transporter elucidates antidepressant mechanism. Nature 503, 85–90 (2013)
Ramamoorthy, S. et al. Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization. Proc. Natl Acad. Sci. USA 90, 2542–2546 (1993)
Green, E. M., Coleman, J. A. & Gouaux, E. Thermostabilization of the human serotonin transporter in an antidepressant-bound conformation. PLoS ONE 10, e0145688 (2015)
Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)
Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nature Protocols 9, 2574–2585 (2014)
Krishnamurthy, H. & Gouaux, E. X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature 481, 469–474 (2012)
Chen, J. G., Liu-Chen, S. & Rudnick, G. External cysteine residues in the serotonin transporter. Biochemistry 36, 1479–1486 (1997)
Tate, C. G. & Blakely, R. D. The effect of N-linked glycosylation on activity of the Na+- and Cl−-dependent serotonin transporter expressed using recombinant baculovirus in insect cells. J. Biol. Chem. 269, 26303–26310 (1994)
Kilic, F. & Rudnick, G. Oligomerization of serotonin transporter and its functional consequences. Proc. Natl Acad. Sci. USA 97, 3106–3111 (2000)
Rasmussen, T. N., Plenge, P., Bay, T., Egebjerg, J. & Gether, U. A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation. Neuropharmacology 57, 287–294 (2009)
Owens, M. J., Knight, D. L. & Nemeroff, C. B. Second-generation SSRIs: human monoamine transporter binding profile of escitalopram and R-fluoxetine. Biol. Psychiatry 50, 345–350 (2001)
Andersen, J. et al. Mutational mapping and modeling of the binding site for (S)-citalopram in the human serotonin transporter. J. Biol. Chem. 285, 2051–2063 (2010)
Koldsø, H. et al. The two enantiomers of citalopram bind to the human serotonin transporter in reversed orientations. J. Am. Chem. Soc. 132, 1311–1322 (2010)
Sørensen, L. et al. Interaction of antidepressants with the serotonin and norepinephrine transporters: mutational studies of the S1 substrate binding pocket. J. Biol. Chem. 287, 43694–43707 (2012)
Barker, E. L. et al. High affinity recognition of serotonin transporter antagonists defined by species-scanning mutagenesis. An aromatic residue in transmembrane domain I dictates species-selective recognition of citalopram and mazindol. J. Biol. Chem. 273, 19459–19468 (1998)
Forrest, L. R., Tavoulari, S., Zhang, Y. W., Rudnick, G. & Honig, B. Identification of a chloride ion binding site in Na+/Cl−-dependent transporters. Proc. Natl Acad. Sci. USA 104, 12761–12766 (2007)
Kantcheva, A. K. et al. Chloride binding site of neurotransmitter sodium symporters. Proc. Natl Acad. Sci. USA 110, 8489–8494 (2013)
Humphreys, C. J., Wall, S. C. & Rudnick, G. Ligand binding to the serotonin transporter: equilibria, kinetics, and ion dependence. Biochemistry 33, 9118–9125 (1994)
Andersen, J. et al. Molecular determinants for selective recognition of antidepressants in the human serotonin and norepinephrine transporters. Proc. Natl Acad. Sci. USA 108, 12137–12142 (2011)
Henry, L. K. et al. Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants. J. Biol. Chem. 281, 2012–2023 (2006)
Tavoulari, S., Forrest, L. R. & Rudnick, G. Fluoxetine (Prozac) binding to serotonin transporter is modulated by chloride and conformational changes. J. Neurosci. 29, 9635–9643 (2009)
Zheng, H., Chruszcz, M., Lasota, P., Lebioda, L. & Minor, W. Data mining of metal ion environments present in protein structures. J. Inorg. Biochem. 102, 1765–1776 (2008)
Singh, S. K., Yamashita, A. & Gouaux, E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448, 952–956 (2007)
Zhou, Z. et al. LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake. Science 317, 1390–1393 (2007)
Zhou, Z. et al. Antidepressant specificity of serotonin transporter suggested by three LeuT-SSRI structures. Nature Struct. Mol. Biol. 16, 652–657 (2009)
Plenge, P. et al. Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter. J. Biol. Chem. 287, 39316–39326 (2012)
Jacobsen, J. P. et al. The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse. Psychopharmacology (Berl.) 231, 4527–4540 (2014)
Scanlon, S. M., Williams, D. C. & Schloss, P. Membrane cholesterol modulates serotonin transporter activity. Biochemistry 40, 10507–10513 (2001)
Koban, F. et al. A salt bridge linking the first intracellular loop with the C terminus facilitates the folding of the serotonin transporter. J. Biol. Chem. 290, 13263–13278 (2015)
Sucic, S. et al. Switching the clientele: a lysine residing in the C terminus of the serotonin transporter specifies its preference for the coat protein complex II component SEC24C. J. Biol. Chem. 288, 5330–5341 (2013)
Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W. & Howard, J. C. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266, 550–552 (1977)
Reeves, P. J., Callewaert, N., Contreras, R. & Khorana, H. G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl Acad. Sci. USA 99, 13419–13424 (2002)
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)
Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)
Bunkóczi, G. et al. Phaser.MRage: automated molecular replacement. Acta Crystallogr. D 69, 2276–2286 (2013)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012)
Šali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993)
Kyte, J. & Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132 (1982)
Acknowledgements
We thank D. Cawley for generating monoclonal antibodies and Lundbeck for Br-citalopram. We thank A. Penmatsa and K. Wang for assistance with initial crystal screening, K. Dürr and W. Lü for help with Fab crystallization and structure refinement, respectively, L. Vaskalis for assistance with figures, H. Owen for help with manuscript preparation and other Gouaux laboratory members for discussions. We acknowledge the staff of the Berkeley Center for Structural Biology at the Advanced Light Source and the Northeastern Collaborative Access Team at the Advanced Photon Source for assistance with data collection. J.A.C. has support from a Banting postdoctoral fellowship from the Canadian Institutes of Health Research. We are particularly grateful to Bernie and Jennifer LaCroute for their generous support, as well as for funding from the National Institutes of Health (NIH) (5R37MH070039). E.G. is an Investigator with the Howard Hughes Medical Institute.
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J.A.C., E.M.G. and E.G. designed the project. E.M.G. and J.A.C. developed thermostable constructs for crystallization. J.A.C. performed protein purification, crystallography and biochemical analysis. J.A.C., E.M.G. and E.G. wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Construct design and secondary structure.
Thrombin digestion sites were introduced within the N- and C-terminal regions before Gln76 and after Thr618. Mutations which were introduced to increase thermostability (Tyr110Ala, Ile291Ala and Thr439Ser) are indicated (red star). Surface exposed cysteine residues were mutated to alanine (Cys554 and Cys580) and indicated by a blue star. Residues that have no electron density are boxed in green. Secondary structure was analysed using DSSP (http://swift.cmbi.ru.nl/gv/dssp/) and displayed using ENDScript (http://endscript.ibcp.fr/). Secondary structure elements are shown using the following symbols: α-helix (α), β-strand (β), π-helix (π), 310 helix (η), β-turn (TT letters), α-turn (TTT letters). Locations of carbohydrate (red, ″) and disulfide bonding cysteine (green digits) residues are also shown. A–C in italic means the residue has a crystallographic contact with a residue in chain A–C. The ‘#’ symbol identifies a contact between two residues along the crystallographic two-fold axis of symmetry. Contacts between transporter residues and small molecules in the range of 3.2–5.0 Å are also indicated (black, ″). Hydropathy is calculated according to Kyte and Doolittle59 and shown with pink as hydrophobic (H > 1.5), cyan as hydrophilic (H < 1.5), and grey as intermediate. The secondary structure of the dopamine transporter (4M48) is shown for comparison.
Extended Data Figure 2 Comparison of the ts3 and ts2 structures, crystal packing and antibody structure.
a, Superposition of the ts2 (blue) and ts3 (grey) transporters, each in complex with paroxetine using all atoms (Extended Data Table 3). Paroxetine (pink sticks) and thermostabilizing mutations (yellow spheres). b, Position of amino acid changes due to single nucleotide polymorphisms and mutants associated with psychiatric disorders (yellow). Paroxetine is shown in pink. c, SERT is shown in green, Fab heavy chain (orange), light chain (blue). SERT molecules pack into the crystal lattice with SERT–SERT interface occurring along the kink of TM12 helices related by the crystallographic two-fold axis (blue box). d, Rotation by 90° reveals further lattice contacts. Red box shows interface between Fab, EL2 and EL4. We predict that this interface contains the high-affinity interaction of the Fab with EL2 and EL4. Also shown is an EL2–EL2 interaction between symmetry related molecules as well as a Fab–EL2 interface in the asymmetric unit. Purple box shows interface between Fab variable domains. Black box shows crystal contact between the C-terminal helix and the Fab constant domain. e, The binding site of the 8B6 Fab is made up of interactions of residues from EL2 and EL4 (sticks). f, Comparison of the high resolution Fab structure (grey) with SERT-bound Fab (Extended Data Table 3). The largest structural changes occur in the complementary determining regions (CDRs).
Extended Data Figure 3 Comparison of ligand binding in SERT and in DAT.
a, Comparison of SERT bound to paroxetine with dDAT (4M48) bound to nortriptyline (yellow); superposition based on TM1–TM12. SERT is shown in blue and DAT in grey. b, Alignment of paroxetine (blue) and (S)-citalopram (pink) structures using all atoms in superposition (Extended Data Table 3). Residues interacting with the antidepressant molecules are shown as sticks. Paroxetine (pink) and (S)-citalopram (green) are shown as sticks. c, Insertion of benzodioxol and fluorophenyl groups of paroxetine and (S)-citalopram into a cavity in subsite B made up of Leu443, Ala169, Ala173 and Ser439. Note that Ser439 is equivalent to Thr439 in wild-type SERT. Equivalent residues in dDAT are shown in grey.
Extended Data Figure 4 Ion-binding sites.
a, Overall view of the Na1 and Cl− ion binding sites in the paroxetine bound transporter. Na+ (salmon) and Cl− (green) are shown as spheres. Paroxetine is shown as pink sticks. b, Overall view of the (S)-citalopram bound transporter showing the Na2 binding site; (S)-citalopram (green sticks). c, Residues coordinating Na1 and Cl−. Ion Fo − Fc omit densities are shown at 2σ and 3σ for Na1 and Cl−. d, Residues coordinating Na2. Fo − Fc omit density is shown at 4σ. A water molecule is shown as a yellow sphere. Coordination distances are given in Extended Data Table 4.
Extended Data Figure 5 Extracellular and intracellular gates and the allosteric site of paroxetine and partially occupied (S)-citalopram.
a, The extracellular gate of the SERT–(S)-citalopram complex is shown, with (S)-citalopram bound to the central site. The width of the gate is depicted by the distances between Tyr176 and Phe335 (10.3 Å, CD1–CE2), Asp98 and Tyr176 (4.0 Å, OD2–OH), Glu494 and Arg104 (4.9 Å, OE1–NH1) dDAT (grey) is shown for comparison. b, Comparison of the intracellular gate of SERT (pink) versus DAT (4M48, grey). Superpositions were made by alignment of TM1–TM12 of SERT with dDAT. c, The allosteric site containing fully occupied (S)-citalopram (pink) was superposed with the partially occupied structure (olive). The Fo − Fc omit density (blue mesh) of the partially occupied structure is shown at 2σ. (S)-citalopram is shown in green sticks. A 12-carbon chain (magenta) was modelled into this density but could instead represent a partially occupied (S)-citalopram. The structure with partial (S)-citalopram occupancy at the allosteric site was derived from crystals grown in the presence of 10 μM ligand. Crystals with a higher occupancy at the allosteric site were soaked in a solution containing 5 mM (S)-citalopram before crystal cryo protection. d, The paroxetine-bound transporter contains a maltose detergent headgroup (orange) bound to the allosteric site. Fo − Fc maltose omit density at 3σ.
Extended Data Figure 6 Cholesteryl hemisuccinate and tetradecane binding sites.
a, Overall view of the (S)-citalopram-bound structure showing CHS (red box) and tetradecane (C14, blue box). b, Zoomed view of the CHS binding site. Residues near CHS are shown as sticks. The Fo − Fc omit density map is shown at 3σ. c, Binding of tetradecane. The Fo − Fc omit density map is contoured at 4σ. d, Tetradecane was modelled on a two-fold axis of symmetry with partial occupancy as a single molecule. On the basis of the density, it is unclear if this molecule represents the alkyl chain of a lipid, detergent, or a molecule of PEG.
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Coleman, J., Green, E. & Gouaux, E. X-ray structures and mechanism of the human serotonin transporter. Nature 532, 334–339 (2016). https://doi.org/10.1038/nature17629
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DOI: https://doi.org/10.1038/nature17629
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