Abstract
Noradrenaline, also known as norepinephrine, has a wide range of activities and effects on most brain cell types1. Its reuptake from the synaptic cleft heavily relies on the noradrenaline transporter (NET) located in the presynaptic membrane2. Here we report the cryo-electron microscopy (cryo-EM) structures of the human NET in both its apo state and when bound to substrates or antidepressant drugs, with resolutions ranging from 2.5 Å to 3.5 Å. The two substrates, noradrenaline and dopamine, display a similar binding mode within the central substrate binding site (S1) and within a newly identified extracellular allosteric site (S2). Four distinct antidepressants, namely, atomoxetine, desipramine, bupropion and escitalopram, occupy the S1 site to obstruct substrate transport in distinct conformations. Moreover, a potassium ion was observed within sodium-binding site 1 in the structure of the NET bound to desipramine under the KCl condition. Complemented by structural-guided biochemical analyses, our studies reveal the mechanism of substrate recognition, the alternating access of NET, and elucidate the mode of action of the four antidepressants.
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Data availability
Atomic coordinates of NET in the apo state or in the presence of substrates or antidepressants in various conditions (NET apo, NET–NE, NET–DA, NET–DSP, NET–BPP, NET–ESC, NET–NE-nd, NET–ATX and NET–DSP–KCl) have been deposited in the Protein Data Bank (http://www.rcsb.org) under the accession codes 8HFE, 8HFF, 8HFG, 8HFI, 8HFL, 8I3V, 8WGX, 8Z1L and 8WGR, respectively. The corresponding electron microscopy maps have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/), under the accession codes EMD-34718, EMD-34719, EMD-34720, EMD-34721, EMD-34722, EMD-35157, EMD-37520, EMD-39729 and EMD-37515, respectively. For the molecular dynamics simulations, the starting and structure files for the molecular dynamics simulations are available on Zenodo (https://zenodo.org/records/11261148). Further data that support the findings of this study are available from the corresponding authors on reasonable request.
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Acknowledgements
We thank N. Zhou for technical support during electron microscopy data collection; the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for providing the cryo-EM facility support and the computational facility support. This work was funded by the National Key R&D Program of China (2020YFA0509301 to C.Y.), the National Natural Science Foundation of China (32171204 to C.Y.), the Tsinghua-Peking University Center for Life Sciences (20111770319 to C.Y. and 20111770319 to B.T.), the Tsinghua University Initiative Scientific Research Program (20221080032 and 20231080037 to C.Y., and 20221080048 and 20231080030 to B.T.), the Beijing Frontier Research Center for Biological Structure, the Beijing Advanced Innovation Center for Structural Biology, and start-up funds from the Tsinghua-Peking Center for Life Sciences and the Tsinghua University.
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C.Y. and Y.Y. conceived the project. J.T., Y.X., Y.Y. and C.Y. designed all experiments. J.T., Y.X., Y.Y., Y.L., H.X. and A.Z. performed the experiments. J.T., Y.X., F.K. and J.L. contributed to cryo-EM data collection. F.K. and C.Y. contributed to the structural determination. X.Z. and B.T. contributed to the molecular dynamics simulation. All authors analysed the data and contributed to manuscript preparation. C.Y. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Biochemical characterization of recombinantly expressed hNET.
a, SEC purification of the hNET in the presence of 0.02% (w/v) DDM for NET apo. b, Peak fractions of SEC purification were further examined by Coomassie blue staining of SDS–PAGE. The experiment was repeated independently 3 times with similar results. c-d, Surface plasmon resonance characteristics of NET under NaCl-DDM/CHS, NaCl-DDM, or KCl-DDM conditions, showing the binding affinity of NET for norepinephrine (panel c) or desipramine (panel d). The KD value was determined using Biacore Insight Evaluation Software via affinity analysis. e-f, Real-time response of the SPR against NET under NaCl-DDM/CHS, NaCl-DDM, or KCl-DDM conditions at different ligand concentrations. The interaction between these two ligands and NET is characterized by ‘fast association and fast dissociation’, resulting in a rapid rise of the curve and reaching a plateau. Therefore, affinity analysis was utilized instead of kinetic analysis.
Extended Data Fig. 2 Data processing of different hNET datasets.
a, Representative micrograph and 2D class averages of NET. The experiment was repeated independently 3 times with similar results. b-c, The flowchart for data processing of NET apo, NET-NE, NET-DA, NET-DSP, NET-BPP, NET-ESC, NET-NE-nd, NET-DSP-KCl, and NET-ATX. d-f, Using NET apo as an example to show the local resolution, gold-standard Fourier shell correlation (FSC) curves from the 3D refinement, and angular distribution of the particles used for the final reconstructions.
Extended Data Fig. 3 Representative cryo-EM densities of NET structures.
a, Representative cryo-EM densities for the TM segments of NET. The densities are contoured at 5.2 σ. Cryo-EM density for the TM1a segment of the outward-open NET-ATX is also shown (blue). b, A comparison of the cryo-EM densities in the S2 pocket in NET datasets. In the NET-apo/DSP/BPP/ESC datasets, no density is observed for the substrate in the S2 pocket. However, in the cryo-EM maps of NET-NE and NET-DA, distinct densities corresponding to NE or DA molecules are clearly visible. The position of the S2 site is indicated by white dashed circles. c, The binding sites of Cl− and K+ ion in NET. Left panel: in NET-DSP (150 mM NaCl), only Cl− density was observed; Right panel: in NET-DSP-KCl (150 mM KCl), in addition to the Cl- density, K+ density was clearly observed at the Na1 site.
Extended Data Fig. 4 The binding and transport of NE and DA in NET.
a, Extracellular gate in NET. Water molecules are depicted as cyan spheres. Arg81 and Phe317 are key residues in the extracellular gate. Arg81 forms a cation-π interaction with Phe317, as indicated by the black dashed line. Arg81 is also coordinated by Gln314, Asp475, and surrounding water molecules. Hydrogen bonds are represented by cyan dashed lines. b, The extracellular gating residues in outward-open DA-bound dDAT. The cation-π interaction between Phe319 and Arg52 (corresponding to Phe317 and Arg81 in NET) and the salt bridge between Asp475 and Arg52 (corresponding to Asp473 and Arg81 in NET) is disrupted. c-d, Similar binding mode of NE and DA in the central substrate binding pocket of NET. DA and NE adopt similar orientations in the central pocket. The β-hydroxyl group of NE faces to the opening tunnel without coordination. e, Determination of the kinetic parameters of NET for the transport of NE and DA. The data were fitted using the Michaelis–Menten non-linear fitting method. Curves were calculated from n = 3 biologically independent experiments.
Extended Data Fig. 5 Structural analysis of the ATX- and DSP-bound NET.
a, A comparative binding analysis between NET-ATX and dDAT-RTX. Left panel: Reboxetine in dDAT are colored light gray. The ethoxy group of reboxetine interacts with Ala117 and Phe325 of the dDAT structure, mirroring the placement of atomoxetine’s methyl group in NET. Right panel: Overlay of dDAT and NET shows a subtle backbone shift in residues 322–325 and the unique role of Pro323. b, A comparative binding analysis between NET-ATX and hDAT. Predicted human DAT structure are colored light gray. The predicted AlphaFold2 structure of DAT exhibits similarities to the NET structure with notable substitutions. Ser149 replaces Ala145, and Val324 replaces Ala321. The larger side chain of Val324 may cause a backbone shift of residues 323–326 towards the central pocket by steric effect. c-d, Distinct binding modes of desipramine or its analogs nortriptyline. Upper panel c: Desipramine binds within the central pocket of NET in an inward-open state. Lower panel c: In the inward-occluded LeuT structure, desipramine binds to an allosteric site distinct from the central pocket where leucine and two sodium ions bind. Panel d: Nortriptyline, an analog of desipramine, binds to the central pocket of dDAT in an outward-open state. Its orientation differs from that of desipramine in NET. e, The potential inhibitory mechanism of desipramine on NET. The first DSP binding site is the NTL binding site in the dDAT-NTL of the outward-open conformation; the second one is the DSP binding site in the LeuT-DSP of the occluded conformation; the third one is the DSP binding site in the NET-DSP of the inward-open conformation; and the last one is the DSP-2 binding site in the NET-DSP-KCl. Four distinct desipramine binding sites are marked with different colors: rose red, light blue, orange, and green. The dashed blue line symbolizes a potential transport pathway for desipramine within NET.
Extended Data Fig. 6 Structural analysis of the ESC-bound NET.
a, The escitalopram-bound NET in an inward-open state. b, The escitalopram-bound SERT in an outward-open state. c, Escitalopram binding mode in NET and SERT. The position and orientation of escitalopram in SERT and NET exhibit notable variations. d. The escitalopram binding pocket of NET and SERT in the inward-open conformation. A critical residue, Gly422 in NET (corresponding to Ala441 in SERT), appears to play a pivotal role in determining the inward-open binding site. Specifically, Ala441 in SERT would result in clashes with both escitalopram and talopram, indicating that this pocket for escitalopram or talopram may be predominantly conserved in NET but not in SERT. The cyano-group of escitalopram seems not to be a decisive factor. e, Local conformational change of SERT to accommodate the cyano-group of escitalopram. To accommodate the cyano-group of escitalopram, a rigid shift occurs in Ala496-Thr497-Gly498, causing Gly498 to face with the cyano-group and minimize steric clashes. f, Comparison of the local structure of NET and SERT related to the cyano-group of escitalopram. in this specific local region, NET and SERT show distinct residue compositions (NET residues 476–481: AAGTSI; SERT residues 496–501: ATGPAV;), with four out of six residues differing. The variance in properties between Thr479 in NET and Pro499 in SERT, along with the local hydrogen bond established by Ser480 with the main chain in NET, could hinder the required conformational changes to accommodate the cyano-group.
Extended Data Fig. 7 The S2 pocket in NET.
a, The S1 and S2 binding pocket in NET and SERT. NET contains two distinct binding pockets for NE or DA. One serves as the central substrate binding site, while the other is situated at the C-terminal end of TM1b (left and middle panels). In the case of SERT, the second 5-HT binding site is situated within an aromatic pocket on the extracellular side, which is formed by TM10, TM11, and TM12 (right panel). The binding sites for NE, DA, and 5-HT are indicated by hexagons that are colored purple, cyan, and green, respectively. b, Substrate recognition in the S2 pocket. TM1b, TM7, EL3, and EL4 are colored marine, purple, yellow, and salmon, respectively. The pocket consists of Tyr87 and Lys88 in TM1b, Phe362 in TM7, Glu377 and Glu382 in EL4. The catechol group of the NE or DA molecule is sandwiched by Gly90 from TM1b and Glu377 and Ile376 from EL4. The amino group of NE or DA forms two hydrogen bonds, one with the carboxyl group of Glu382 and the other with the carbonyl group of Lys88. Additionally, Phe362, Ala293, and Tyr87 engage in hydrophobic interactions with the benzene ring of NE or DA. Water-mediated interactions may also occur between the two hydroxyl groups of NE or DA and the surrounding residues Arg301, His296, Asp298, and Glu377. Left panel: the NE molecule in the S2 pocket. The additional β-hydroxyl group of NE forms hydrogen bonds with the carboxyl group of Glu382 and the carbonyl group of Tyr87. Right panel: the DA molecule in the S2 pocket. c, The S2 site in the MATs. Left panel: The S2 site in hNET; Middle panel: The corresponding site in dDAT. Right panel: The corresponding site in hSERT. The A293 residue in hNET, along with its corresponding residues Y295 in dDAT and F311 in hSERT, are highlighted in orange. d, Biochemical validation of substrate binding residues in the S2 site using a proteoliposome-based [3H]-NE uptake assay. Three biological experiments were carried out in the assay: one experiment with three technical repeats and two experiments with two technical repeats (n = 7). The Student’s t-test was used to show significance of mutations compared with WT: ****P < 0.0001 and not significant (NS). Statistical significance was set at P < 0.05. From top to bottom, P = 0.5815, P < 0.0001, P = 0.3626, P < 0.0001.
Extended Data Fig. 8 The charge-dipole interaction of DA with TM6a and the CTH of NET.
a, Recognition of the amino group of DA in NET and dDAT. In addition to a hydrogen bond with Phe317, the amino group of DA can also be attracted by the C-terminal end of the helical dipole TM6a that is negatively charged. b, The CTH of NET. CTH, IL5, IL1, TM10, and TM11 are colored red, yellow, cyan, green, and orange, respectively. The four segments of the CTH are labeled as segments a, b, c, and d. c, Four ion pair interactions are indicated by cyan circles in CTH: Arg512 and Arg518 with the terminal carboxyl group of Ile617, Asp499 with Arg607, Arg500 with Glu597, the carbonyl groups of Trp494 and Phe495 with Gln608 and His603. d, Three hydrophobic interactions are highlighted with light yellow circles: Leu515, Leu519, Phe523 and Ile617; Trp494, Phe495 and Trp614; Trp585, Leu588, Ile592, Ile606 and Phe609.
Extended Data Fig. 9 Characterization of the inhibition type of atomoxetine, desipramine, bupropion, and escitalopram.
a, The [3H]-NE uptake assays by NET in the absence (0 nM in yellow) or presence (30 nM in green and 120 nM in rose red) of atomoxetine. The data were fitted using the Michaelis–Menten non-linear fitting method. Curves were calculated from three biologically independent experiments: one experiment with three technical repeats and two experiments with two technical repeats (mean ± SEM; n = 7). Non-linear regression fits show characteristic results for competitive inhibition of NET by atomoxetine. b-d, The [3H]-NE uptake assays revealed inhibition mechanism for desipramine, bupropion, and escitalopram. The results are illustrated with yellow curves to represent the absence of inhibitors (WT), green curves for medium inhibitor concentration, and rose red curves for high inhibitor concentration. The data were fitted using the Michaelis–Menten non-linear fitting method. Curves were calculated from three biologically independent experiments: one experiment with three technical repeats and two experiments with two technical repeats (mean ± SEM; n = 7). Non-linear regression fits show characteristic results for non-competitive (mixed-type) inhibition of NET by desipramine, bupropion, and escitalopram.
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Tan, J., Xiao, Y., Kong, F. et al. Molecular basis of human noradrenaline transporter reuptake and inhibition. Nature 632, 921–929 (2024). https://doi.org/10.1038/s41586-024-07719-z
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DOI: https://doi.org/10.1038/s41586-024-07719-z
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