Abstract
The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons1,2,3. It is a pharmacological target for various antidepressants and analgesic drugs4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl− undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.
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Data availability
The three-dimensional cryo-EM density maps of the NETApo, NETNA, NETMrlA, NETZPD and NETBPP have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-37842, EMD-37843, EMD-37844, EMD-37845 and EMD-37846, respectively. The coordinates for NETApo, NETNA, NETMrlA, NETZPD and NETBPP have been deposited in Protein Data Bank under accession codes 8WTU, 8WTV, 8WTW, 8WTX and 8WTY, respectively. Source data are provided with this paper.
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Acknowledgements
We thank X. Huang, B. Zhu, X. Li, L. Chen and other staff members at the Center for Biological Imaging (CBI), Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Science (IBP, CAS) for the support in cryo-EM data collection; H. Zhang and T. Sun for their assistance in the [3H]noradrenaline uptake assays; Y. Chen, Z. Yang and B. Zhou for technical help with Biacore experiments; NanoTemper Technologies China for assistance with spectral shift assays; Y. Wu for his research assistant services; and members of the Zhao laboratory for helpful discussions. This work is funded by the Chinese Academy of Sciences Strategic Priority Research Program (grant no. XDB37030304 to Y.Z.), Chinese National Programs for Brain Science and Brain-like Intelligence Technology (grant no. 2022ZD0205800 to Y.Z.), the National Key Research and Development Program of China (grant no. 2021YFA1301501 to Y.Z.) and the National Natural Science Foundation of China (grant no. 92157102 to Y.Z.).
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Contributions
Y.Z. conceived and supervised the project. T.H. carried out molecular cloning experiments, expressed and purified protein samples, and prepared samples for cryo-EM study. T.H., Y.M, K.S., C.J.L., Y.W. and B.Y. performed functional assays. J. Zhang, S.X., Q.D. and R.Y. synthesized the conopeptides. Z.Y., J.Z. and T.H. carried out cryo-EM data collection. Z.Y. processed the cryo-EM data. Z.Y., T.H. and Q.B. built the atomic model and analysed the structures. Z.Y. and T.H. prepared the figures. Y.Z., T.H., C.J.L. and Z.Y. wrote and revised the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Purification and functional characterization of NET.
a-b, Gel-filtration profile of purified NETWT (a) and NETEM (b) in the presence of 0.025% (w/v) DDM plus 0.015% CHS. c, Concentration-dependence of norepinephrine uptake by NETWT and NETEM. Data represent mean ± SEM obtained from three biological experiments. There is no significant difference (p = 0.4) in Km value for NETEM (0.32 ± 0.05μM) and NETWT (0.24 ± 0.03μM). d-f, Inhibition of [3H]NE uptake in NETEM and NETWT by χ-MrlAEM (d), bupropion (e), and ziprasidone (f). Data represent mean ± SEM obtained from three biological experiments. t-test was employed to compare the IC50 values for χ-MrlAEM, bupropion, and ziprasidone between NETEM and NETWT. The resulting p-values were 0.0916, 0.7056, and 0.0541, respectively, indicating that the differences were not statistically significant. g, Gel-filtration profile of NETEM in nanodisc. h, Coomassie-blue-stained SDS-PAGE gel of NETEM in nanodisc. For gel source data, see Supplementary Fig. 1. i, Concentration-dependence of norepinephrine uptake of NETEM in proteoliposome. The results were fitted to ‘Michaelis Menten’ equation, determining the Km and Vmax value of norepinephrine transport as 0.17 ± 0.08 μM and 904.7 pmol/min/mg, respectively. j-l, Inhibition of [3H]NE uptake by χ-MrlAEM (j), bupropion (k), and ziprasidone (l) on NETEM in proteoliposome. The normalized data of inhibition assay were fitted using non-parametric nonlinear regression, yielding IC50 values of 113.5 ± 34.2 nM for χ-MrlAEM, 6.0 ± 3.1 μM for bupropion and 259.4 ± 87.1 nM for ziprasidone. m-p, Competition binding assays for nisoxetine (m), χ-MrlAEM (n), bupropion (o), and ziprasidone (p) were performed to compete against [3H]nisoxetine using scintillation proximity assay. Data points represent mean ± SEM from three independent assays. The Ki values for nisoxetine, χ-MrlAEM, bupropion, and ziprasidone are 21.9 ± 4.7 nM, 144.3 ± 11.3 nM, 13.1 ± 1.4 μM, and 163.7 ± 20.3 nM, respectively.
Extended Data Fig. 2 Cryo-EM reconstruction of NETApo.
a, Cryo-EM data processing workflow for NETApo in the inward-open conformation. A representative micrograph was shown with the white bar equals 100 nm. b, Local resolution map of NETApo, with colors representing local resolution estimates ranging from 2.0 to 3.0 Å. c, Angular sampling of the final reconstruction, illustrating the orientation distribution of particles in the final reconstruction of NETNA. d, Map-map and map-model Fourier shell correlations (FSC) curves. e, Electron density visualization of transmembrane helices in the NETApo structure.
Extended Data Fig. 3 C-terminus structure of NET.
(left): The cartoon representation of the NETMrlA structure is color-coded in a rainbow spectrum. (middle): Detailed view of c-terminus of NET. The electron density is depicted as a slate-colored mesh. Residues within the C-terminus are visualized as sticks. (right): Interaction network between c-terminus and core region of NET. Residues participating in these interactions are represented as sticks and labeled. Hydrogen bond interactions are represented as black dash lines, with distances labeled.
Extended Data Fig. 4 Structural analysis of the norepinephrine binding sites.
a-d, Cryo-EM density (blue mesh) corresponding to substrate norepinephrine at central binding site (a/c) and S2 binding site (b/d) in the NETNA structure and NETApo structure, respectively. The density maps are showed in threshold countered at 1.0 (left in a), 0.6 (right in a), 0.8 (b), 0.6 (c) and 0.8 (d) in ChimeraX, respectively. NETNA is shown as cyan cartoon, and substrate NE is shown as orange sticks. e, Overall structural comparison of NETApo (cyan) and NETNA (orange). f, Detailed comparison of S1 binding site between NETNA and NETApo. Residues surrounding NA are shown as sticks. g, Detailed comparison of S2 binding site between NETNA and NETApo. Residues surrounding NA are shown as sticks. h, The sequence alignment of NET from different species at key residues in the NE binding sites. i, Structural alignment with SERT (PDB: 7LI9) using overall structure. Superposition of NET (orange) onto SERT (skyblue) complexed with serotonin, both adopting the inward conformation. S2SERT and S2NET: Secondary binding sites specific to 5-HT and NA, respectively.
Extended Data Fig. 5 Amino acid sequence of human monoamine transporter.
Multiple sequence alignment analysis was conducted for monoamine transporters, incorporating human NET (Uniprot ID: P23975), SERT (Uniprot ID: P31645), and DAT (Uniprot ID: Q01959). Strictly conserved negatively and positively charged residues were respectively highlighted in red and blue, while other conserved residues were annotated in grey. Black bars above the sequence were employed to indicate every ten residues, including gaps. The transmembrane helices were labeled with blue strips. The orange and light green hexagon show residues involved in coordinating substrate in S1 and S2 substrate binding site, respectively. The green stars indicate residues involved in mutation studies of χ-MrlA. The yellow and magenta circles mark residues involved in mutation studies of bupropion and ziprasidone, respectively.
Extended Data Fig. 6 Structural analysis of χ-MrlAEM and ion binding sites.
a, HPLC chromatogram of MrlAEM obtained using the Waters e2695 Separations Module and 2998 PDA Detector at 220 nm, the mobile phase employed a 30-minute gradient from 90% mobile phase A to 60% mobile phase A (mobile phase A: water with 0.05% TFA; mobile phase B: acetonitrile with 0.05% TFA). b, MS chromatogram of MrlAEM using the Waters 2998 PDA Detector, the theoretical mass of MrIAEM was 1660.03, and the observed mass-to-charge ratio (m/z) were as follows: 830.35[M + 2H]2 +, 553.97[M + 3H]3 +, 415.64[M + 4H]4 +. c, Chemical structure of MrlAEM drawn by ChemBioDraw Ultra 14.0, the unnatural amino acid MeY and O represents O-methyl-L-tyrosine and L-trans-hydroxyproline, respectively. d, Superposition of χ-MrlAEM (magenta) and χ-MrlA (PDB ID: 2EW4) (green) are based on the overall structure. The one-letter code and position of the corresponding amino acids are labeled. MeY represents o-methyl-l-tyrosine. e-f, Superposition between NETMrlA (pink) and NETNA (orange) based on scaffold domain. The binding positions of χ-MrlA and NA do not overlap while χ-MrlA and NA are represented as green and orange sticks/spheres, respectively. g-h, Superposition between NETMrlA (pink) and dDATNisoxetine (PDB ID: 4XNU) (yellow) based on scaffold domain. The binding positions of χ-MrlA and Nisoxetine overlap while χ-MrlA and Nisoxetine are represented as green and purple sticks/spheres, respectively. i, Interaction network at the ionic binding site in the outward-open structure NETMrlA. Sodium and chloride ions are represented by purple and green balls, respectively. j, Interaction network at the ionic binding site in the inward-open structure NETNA. Sodium and chloride ions are represented by brown and yellow balls, respectively.
Extended Data Fig. 7 Structural comparisons of substrate and inhibitor binding sites.
a, The displacement of substrate NA during conformational transition from outward-facing to inward-facing state. Structural comparison of NETNA and dDATNA (PDB: 6M0Z) are carried out using the scaffold domain as a reference. The structures of NETNA and dDATNE are colored in orange and grey, respectively. (inset) Substrate NA molecules are shown as spheres and sticks. Key residues involved in substrate binding are represented as sticks. The displacement of substrate NA and side chains of key residues are labeled. b, Binding position comparison of bupropion, ziprasidone and ibogaine. The structures of NETBPP and NETZPD are depicted as slate and green cartoon, respectively. The structure of SERT bound with ibogaine (SERTibogaine) in inward-facing state (PDB ID: 6DZZ) is depicted as green cartoon. Ligands are represented as spheres. c, The binding mode of bupropion. Superposition was performed between NETBPP and SERT bound with serotonin (SERT5-HT) in inward-facing state (PDB ID: 7LI9) based on the overall structure. NETBPP and SERT5-HT are colored in blue and grey, respectively. Residues surrounding the binding site are visualized as sticks. d, The binding mode of ziprasidone. Superposition was carried out between NETZPD and SERT5-HT in the inward-open conformation based on the overall structure. NETZPD and SERT5-HT are colored in green and grey, respectively. Residues surrounding the binding site are visualized as sticks. e, A comparative analysis of the binding modes of bupropion, ziprasidone, and ibogaine is provided, highlighting their differences of binding positions.
Extended Data Fig. 8 The working and inhibitory mechanism of NET.
Key helices involved in the conformational changes are colored and labeled, whereas the scaffold domain and C-terminus are depicted in gray. The intracellular and extracellular cavities are represented by purple shadows. Molecules of noradrenaline, nisoxetine, bupropion, and ziprasidone molecules are depicted as spheres, while χ-conotoxin MrlA is shown as a green cartoon and surface.
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Hu, T., Yu, Z., Zhao, J. et al. Transport and inhibition mechanisms of the human noradrenaline transporter. Nature 632, 930–937 (2024). https://doi.org/10.1038/s41586-024-07638-z
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DOI: https://doi.org/10.1038/s41586-024-07638-z
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