Abstract
The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology1. Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder2. Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders.
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Data availability
The density maps and structure coordinates have been deposited to the Electron Microscopy Data Bank and the PDB under accession numbers EMD-39069 and 8Y94 (NET-apo complex); EMD-39533 and 8YR2 (NET–nisoxetine complex); EMD-39070 and 8Y95 (NET–NE complex); EMD-39067 and 8Y92 (NET–atomoxetine complex); EMD-39064 and 8Y8Z (NET–maprotiline complex); EMD-39068 and 8Y93 (NET–amitriptyline complex); EMD-39065 and 8Y90 (NET–nefopam complex); and EMD-39066 and 8Y91 (NET–nomifensine complex). PDB accessions 5I74, 4M48, 4XP1, 7LIA, 4XNU, 6M0Z, 5I6X, 3F3E, 6KKT and 6M1D were used for structure analysis. Source data are provided with this paper.
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Acknowledgements
We thank Z. Wang, Z. Meng and D. Wang for their collaborative efforts; X. Huang, Y. Li and X. Yang for their assistance with the purification, certification and cryo-EM data processing of NET proteins; H. Li for providing the 293GnTi− cell line; and Y. Liu for supplying the freestyle medium. The Biacore binding test was performed with support from ShanghaiTech University and guidance from Y. Zhang. The cryo-EM data were collected at the Advanced Center for Electron Microscopy, Shanghai Institute of Materia Medica. We thank all staff at the institution for their assistance with cryo-EM data collection. This work was partially supported by the Lingang Laboratory (grant no. LG-GG-202204-01 to Y.J. and H.E.X.); the National Natural Science Foundation (32171187 to Y.J.; 82121005 to Y.J., H.E.X. and D.Y.; 32130022 to H.E.X.; and 82273985 to D.Y.); the CAS Strategic Priority Research Program (XDB37030103 to H.E.X.); the Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X.); the National Key Basic Research Program of China (2023YFA1800804 to D.Y.); the Shanghai Municipality Science and Technology Development Fund (21JC1401600 to D.Y.); the Program of Shanghai Academic/Technology Research Leader (23XD1400900 to D.Y.); the Hainan Provincial Major Science and Technology Project (ZDKJ2021028 to D.Y.); the China Postdoctoral Science Foundation (2023M731487 to Y.-L.Y.); the Shanghai Post-doctoral Excellence Program (2023018 to Y.-L.Y. and 2022232 to B.P.); and the Shanghai Sailing Program (23YF1460700 to Y.-L.Y. and 22YF1461200 to B.P.).
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Contributions
H.Z. designed the expression constructs, purified the NET protein samples, prepared cryo-EM grids, calculated cryo-EM data, built and refined structural models and prepared figures. Y.-L.Y. performed the dimerization assay. Y.-L.Y. and T.Z. performed the neurotransmitter transporter uptake assay and prepared figures. A.D. and C.Z. performed the radiolabelled-ligand-binding study and prepared the corresponding figures. B.P. and S.J. participated in functional studies and data analysis. C.W. participated in cryo-EM data calculation. W.H. and Q.Y. collected cryo-EM data. X.H. analysed structures. D.Y. and M.-W.W. supervised the radiolabelled-ligand-binding study. H.E.X. supervised the project, analysed the structures and modified the manuscript. Y.J. initiated collaborations with H.E.X. and D.Y., supervised the project and wrote the manuscript with inputs from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Effects of NET mutations on the binding affinity of NE and antidepressants.
a, Binding of nisoxetine to NET assembled in nanodiscs containing MSP1D1 and NET expressed on cell membranes of HEK293 cells (whole-cell) in competition with [3H]-nisoxetine. Assays were conducted with four independent biological replicates (n = 4). The unpaired two-tailed t-test was used to determine the statistical difference (P = 0.0533). b, Binding of six depressants in competition with [3H]-nisoxetine. Binding data were analysed using a three-parameter logistic equation to determine pIC50 values. Data were generated and graphed as means ± s.e.m. of three independent biological replicates (n = 3), except for WT for nisoxetine, WT and S420A for maprotiline, which were performed with four independent biological replicates (n = 4).
Extended Data Fig. 2 Cryo-EM structural determination of human NET in the apo state and bound to the native substrate NE or to inhibitors.
a–e, Overall structures of atomoxetine-bound (a), maprotiline-bound (b), apo (c), NE-bound (d) and nomifensine-bound (e) NET dimers. The representative density maps of TM1, TM6, apo NET, and atomoxetine/maprotiline/NE/nomifensine-bound NET dimers are shown. f,g, The two protomers of the NET–amitriptyline (f) and NET–nefopam (g) dimer are asymmetric. The protomer containing amitriptyline or nefopam shows an outward-open state and exhibits better density, whereas the other protomer adopts an occluded conformation and lacks the density of inhibitors.
Extended Data Fig. 3 Representative EM densities of NET.
a, Nisoxetine–NET dimer transmembrane helices and EM densities at a contour level of 0.30. b, Nisoxetine and EM densities at a contour level of 0.32. c, Cholesterols surround NET except for the dimer interface, EM densities at a contour level of 0.18. d, Lipid and cholesterols at the upper layer dimer interface, EM densities at a contour level of 0.20. e, Lipid and cholesterols at the lower layer dimer interface, EM densities at a contour level of 0.20.
Extended Data Fig. 4 Structural comparisons of NET with SERT and dDAT in outward-open states.
a, Structural comparisons of the nisoxetine–NET, paroxetine–SERT (PDB: 5I6X) and nortriptyline–dDAT (PDB: 4M48). The structures were superimposed onto NET (residues P55–E597) and its homologues. b, Structural comparisons of the extracellular segments of TMs 10–12 in NET, SERT and dDAT. The directional movements of TMs 10–12 in NET relative to that in SERT are indicated by black arrows. c, Comparisons of EL2, EL4 and EL6 among NET, SERT and dDAT. The longer EL2 of NET relative to SERT and dDAT protrudes towards EL4, as indicated by the black arrow. d, The NET structure shows an extended C terminus relative to SERT and dDAT. This extended C terminus contacts the C-terminal latch, the cytoplasmic ends of TM10 and TM11, and IL5, thus sealing the cytoplasmic ends of TMs 10–12.
Extended Data Fig. 5 Effects of alanine mutations on the transport activity of NET in response to NE and antidepressants.
a–c, The mutated residues in subsite A (a), subsite B (b) and subsite C (c) of NET were tested. Data were normalized to the response of wild-type NET. The assays were conducted with three independent biological replicates (n = 3). Each data point presents mean ± s.e.m.
Extended Data Fig. 6 Comparisons of the recognition of substrates and antidepressants for NET and its homologues.
a, Sequence alignment of residues in TM1, TM3, TM6 and TM8 that constitute the substrate- or inhibitor-binding pocket of NET. b–d, Structural comparisons of NE–NET with DA–dDAT (b; PDB: 4XP1), 5-HT–SERT (c; PDB: 7LIA) and NE–dDAT (NET-like) (d; PDB: 6M0Z). e, Comparison of binding poses for nisoxetine bound to NET and dDAT (PDB: 4XNU). f,g, Comparison of the binding pose of nisoxetine in NET and citalopram in SERT (f; PDB: 5I74), as well as amitriptyline in NET and nortriptyline in dDAT (g; PDB: 4M48).
Extended Data Fig. 7 Antidepressant selectivity for MATs.
a, The non-conserved residues in the central ligand-binding site among MATs. The SERT-like (F72Y/V148I/G149A) and DAT-like mutations of NET (A145S/Y151F) were highlighted in red. b, Effects of SERT-like NET mutations on the binding affinity of maprotiline and nomifensine. Data were generated and graphed as means ± s.e.m. of three independent biological replicates (n = 3). c, Effects of DAT-like NET mutations on the binding affinity of nisoxetine, atomoxetine, amitriptyline and nefopam. Data were generated and graphed as means ± s.e.m. of three independent biological replicates (n = 3), except for WT and Y151F for amitriptyline, which were performed in five independent biological replicates (n = 5). d, Structure superposition of maprotiline- and nomifensine-bound NET. e, Structure superposition of nisoxetine-, atomoxetine-, amitriptyline- and nefopam-bound NET.
Extended Data Fig. 8 The unique homodimer of NET.
a–c, Homodimer interfaces of transporters with reported structures, including bacterial leucine transporter (LeuT) (a; PDB: 3F3E), solute carrier family 12 (potassium/chloride transporter) member 4 (SLC12A4) (b; PDB: 6KKT) and sodium-dependent neutral amino acid transporter B(0)AT1 (solute carrier family 6 member 19; SLC6A19) (c; PDB: 6M1D). d, Sequence alignment of residues in TM3, TM4, TM9 and TM12 at the homodimer interface of NET. e, Sequence conservation between three monoamine transporters (NET, DAT and SERT) in cartoon representation. Conservation is coloured as indicated. Higher numbers denote higher conservation.
Extended Data Fig. 9 Binding details of cholesterols and lipids with residues at the homodimer interface of NET.
a, The two PIs (phosphatidylinositol) and six cholesterol molecules (CLRs) located at the upper layer interface from the extracellular view. b, The six cholesterols, two PIs, two phosphatidylinositol 4,5-bisphosphate (PIP2) packed at the lower layer interface from the bottom view. c–h, Detailed interactions between specific CLR or lipid with residues at the homodimer interface of NET are as follows: CLRs and PI (c), PI #1 and CLR #2 (d), CLR #1 and CLR #3 located in the upper layer (e), as well as CLRs, PI and PIP2 (f), PI #2 and CLR #4 (g), CLR #5 and PIP2 located in the lower layer (h). Cholesterol (dodger blue), PI (medium purple), and PIP2 (deep sky blue) are represented in ball-and-stick format (blue). The side chains of residues in two protomers of NET (cyan and purple) are shown with sticks.
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Zhang, H., Yin, YL., Dai, A. et al. Dimerization and antidepressant recognition at noradrenaline transporter. Nature (2024). https://doi.org/10.1038/s41586-024-07437-6
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DOI: https://doi.org/10.1038/s41586-024-07437-6
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