ABSTRACT Norepinephrine transporter (NET; encoded by _SLC6A2_) reuptakes the majority of the released noradrenaline back to the presynaptic terminals, thereby affecting the synaptic

noradrenaline level1. Genetic mutations and dysregulation of NET are associated with a spectrum of neurological conditions in humans, making NET an important therapeutic target1. However,

the structure and mechanism of NET remain unclear. Here we provide cryogenic electron microscopy structures of the human NET (hNET) in three functional states—the apo state, and in states

bound to the substrate meta-iodobenzylguanidine (MIBG) or the orthosteric inhibitor radafaxine. These structures were captured in an inward-facing conformation, with a tightly sealed

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MECHANISMS OF THE HUMAN NORADRENALINE TRANSPORTER Article 31 July 2024 MOLECULAR BASIS OF HUMAN NORADRENALINE TRANSPORTER REUPTAKE AND INHIBITION Article 24 July 2024 TRANSPORT AND

INHIBITION MECHANISM FOR VMAT2-MEDIATED SYNAPTIC VESICLE LOADING OF MONOAMINES Article Open access 02 January 2024 DATA AVAILABILITY Cryo-EM maps of hNET in the apo state, the MIBG-bound

state and the radafaxine-bound state have been deposited in the Electron Microscopy Data Bank under accession codes EMD-38210, EMD-38209 and EMD-38208, respectively. Atomic models of hNET in

the apo state, the MIBG-bound state and the radafaxine-bound state have been deposited at the PDB under accession codes 8XB4, 8XB3 and 8XB2, respectively. The entries 7Y7W, 4XP1, 6M2R,

8GNK, 7SK2, 6ZBV, 6DZZ, 7Y7Y and 6M0F used in this study were downloaded from the PDB. MD simulations (initial coordinate and simulation input files and coordinate files of the final output)

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binding and inhibition mechanism of norepinephrine transporter’. figshare https://doi.org/10.6084/m9.figshare.26043313.v1 (2024). Download references ACKNOWLEDGEMENTS We thank all of the

members of the J.-X.W. laboratory for help; L. Liu for the help with small molecules; X. Wang for the help with docking; the members of the P. Li and B. Cui laboratories for the AKTA

purification platform; the members of the R. Wang laboratory for experimental equipment; the staff at the Biological Analysis Center at Institute of Materia Medica of Chinese Academy of

Medical Sciences for the centrifuge experimental platform; and the staff at Shuimu BioSciences for cryo-EM facility access and technical support during image acquisition. Cryo-EM data

collection was supported by the Electron microscopy laboratory, Cryo-EM platform of Peking University and Shuimu BioSciences with the assistance of X. Li, Z. Guo, C. Qin, X. Pei, X. Hui and

G. Wang. Part of the structural computation was also performed on the Computing Platform of the Center for Life Science and High-performance Computing Platform of Peking University. The work

is supported by grants from National Natural Science Foundation of China (32371266 to J.-X.W.) and the non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (no.

2022-RC350-01 to J.-X.W.). AUTHOR INFORMATION Author notes * These authors contributed equally: Wenming Ji, Anran Miao, Kai Liang, Jiameng Liu AUTHORS AND AFFILIATIONS * State Key

Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P. R. China

Wenming Ji, Anran Miao, Kai Liang, Jiameng Liu, Yuhan Qi, Yue Zhou & Jing-Xiang Wu * Beijing Jingtai Technology, Beijing, P. R. China Xinli Duan, Jixue Sun & Lipeng Lai Authors *

Wenming Ji View author publications You can also search for this author inPubMed Google Scholar * Anran Miao View author publications You can also search for this author inPubMed Google

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inPubMed Google Scholar * Xinli Duan View author publications You can also search for this author inPubMed Google Scholar * Jixue Sun View author publications You can also search for this

author inPubMed Google Scholar * Lipeng Lai View author publications You can also search for this author inPubMed Google Scholar * Jing-Xiang Wu View author publications You can also search

for this author inPubMed Google Scholar CONTRIBUTIONS J.-X.W. initiated the project. A.M., W.J. and Y.Z. screened the expression constructs. W.J. and Y.Q. purified proteins. W.J. prepared

the cryo-EM sample and screened the cryo-EM sample. W.J., A.M., K.L. and J.-X.W. collected the cryo-EM data. J.-X.W. processed the cryo-EM data. J.-X.W. and K.L. built and refined the atomic

model. W.J., K.L., J.L. and Y.Q. performed the uptake assay and surface labelling. X.D., J.S. and L.L. performed the computational analysis on radafaxine binding. All of the authors

contributed to the manuscript preparation. CORRESPONDING AUTHOR Correspondence to Jing-Xiang Wu. ETHICS DECLARATIONS COMPETING INTERESTS X.D., J.S. and L.L. are employees of Beijing Jingtai

Technology. The other authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature_ thanks Gary Rudnick and the other, anonymous, reviewer(s) for their contribution to

the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIG. 1 DESIGN AND PURIFICATION OF HNETCRYO-EM. A. The construct schematic with PA tag insertion in red triangle, amino acid substitutions

(marked above), and GFP-MBP tags (dashed boxes). Predicted glycosylated residues (N192, N198) in EL2-PA tag are in cyan hexagons. B. Structures of norepinephrine (NE), 4-(4-(dimethylamino)

styryl)-N-methylpyridinium iodide (ASP+), meta-iodobenzylguanidine (MIBG), and inhibitor radafaxine. NE and substrate analogues are in orange rectangle. C. Dose-dependent inhibition of

radafaxine on hNETwt. Data are mean ± s.d., n = 4. D. The FSEC profile of hNETwt, hNETdN53, hNETdC18, and hNETcryo-EM. The detection channel is emission (Em): 488 nm, excitation (Ex): 520 

nm. E. SDS-PAGE gel shows properties (glycosylation) of hNETwt, hNETdN53, hNETdC18, and hNETcryo-EM. For gel source data, see Supplementary Fig. 1. F. Interaction between hNETcryo-EM and NZ1

Fv-clasp detected using Native-PAGE. For gel source data, see Supplementary Fig. 1. G. The FSEC profile of hNETcryo-EM alone and in complex with NZ1 Fv-clasp. The detection channel is Em:

488 nm, Ex: 520 nm. H. Normalized ASP+ uptake activity and inhibitory activity by MIBG (200 μM) and radafaxine (200 μM) to hNETwt, hNETcryo-EM, and hNETcryo-EM–NZ1 Fv-clasp complex with

empty vector as negative control. Data are mean ± s.d., n-6. I. Un-normalized uptake assay with inhibitors of H. Data are mean ± s.d., n = 6. J. NET surface labelling to hNETwt and

hNETcryo-EM. Data are mean ± s.d. (Empty vector n = 7; hNETwt n = 8; hNETcryo-EM n = 10). K. NZ1 Fv-clasp surface labelling to hNETcryo-EM alone and hNETcryo-EM–NZ1 Fv-clasp complex. Data

are mean ± s.d. (Empty vector: -NZ1 Fv-clasp n = 7 and +Fv-clasp n = 8; hNETcryo-EM: -NZ1 Fv-clasp n = 8 and +Fv-clasp n = 10). L. SEC profile of hNETcryo-EM -NZ1 Fv-clasp complex in

presence of MIBG, and SDS-PAGE gel of corresponding SEC fractions. For gel source data, see Supplementary Fig. 1. Source Data EXTENDED DATA FIG. 2 CRYO-EM IMAGE ANALYSIS OF HNETCRYO-EM IN

THE APO STATE. A. Representative raw micrograph (4,014 in total) of hNETcryo-EM in the apo state. B. Representative 2D class averages of hNETcryo-EM. C. The cryo-EM data processing workflow

for particle picking, 2D classification, and 3D classification to reach a final three-dimensional reconstruction of hNETcryo-EM at 2.9 Å. D. Angular distribution of all particle images that

contributed to the final 3D map, calculated in cryoSPARC. E. Fourier shell correlation (FSC), indicating the resolution at the 0.143 threshold of unmasked, loosen, tighten, corrected curves

of the map. F. Local resolution distribution of the map of hNETcryo-EM. EXTENDED DATA FIG. 3 LOCAL DENSITY OF HNET IN THE APO STATE. A. Representative densities of transmembrane helices and

C-terminal helices of hNET (54–617). The density of each helix is shown with different map levels: TM5 and CH1-CH3 are contoured at 0.6 level (4.73 σ) and others at 1.2 level (9.47σ). B.

Representative densities of EL2, shown at 0.6 level (4.73 σ). Density is observed for the disulfide bond formed between C176 and C185 at the EL2, shown at 1.2 level (9.47σ). Sticks coloured

in yellow show the disulfide bond interactions. C. The local electron density of hNET at the substrate binding site. D. Electron density of hNET and GAT1 (PDB: 7Y7W) at the Na2 binding site.

E. Un-normalized uptake activities of hNET mutations related to the EC-gate in Fig. 2d. Data are mean ± s.d. (Empty vector n = 25 and others are consistent with Fig. 2d). F. NET surface

labelling of hNET mutations related to the EC-gate in Fig. 2d. Data are mean ± s.d. (Empty vector n = 17; hNETwt n = 18; W80A and R81D n = 9; R81A, R81Q, and T313A n = 7; Q314A, D473A,

D473R, Y152A, and F317A n = 6). G. Un-normalized uptake activities of hNET mutations related to the Na2 binding site in Fig. 2f. Data are mean ± s.d. (n are consistent with Fig. 2f). H. NET

surface labelling of hNET mutations related to the Na2 binding site in Fig. 2f (n = 6). Source Data EXTENDED DATA FIG. 4 SEQUENCE ALIGNMENT. The sequences of (Homo sapiens) hNETcryo-EM,

(Homo sapiens) hNETwt, (Drosophila melanogaster) dmDATmfc (PDB ID: 4XP1), (Drosophila melanogaster) dmDATwt, (Homo sapiens) hDAT, (Homo sapiens) hSERT, and (Homo sapiens) hGAT were aligned

using Jalview. The cylinder represents the helix, and the colour of the cylinder is the same with that in Fig. 1c. The dotted line shows the unmodelled residues. The red box is the deleted

or replaced EL2 residues. The blue boxes are N/C-terminal truncations in the available structures of MATs. The inserted PA tag is displayed in green. The glycosylation sites of NET (N182,

N194, N198) and DAT (N181, N188, N205) are marked with hexagons. EXTENDED DATA FIG. 5 CRYO-EM IMAGE ANALYSIS OF HNETCRYO-EM IN COMPLEX WITH MIBG. A. Representative raw micrograph (4,113 in

total) of hNETcryo-EM binding with MIBG. B. 2D class averages of hNETcryo-EM binding with MIBG. C. The cryo-EM data processing workflow for particle picking, 2D classification, and 3D

classification to reach a final three-dimensional reconstruction of hNETcryo-EM binding with MIBG at 2.8 Å. D. Angular distribution of all particle images that contributed to the final 3D

map, calculated in cryoSPARC. E. Local resolution distribution of the map of hNETcryo-EM binding with MIBG. F. Fourier shell correlation (FSC), indicating the resolution at the 0.143

threshold of unmasked, loosen, tighten, corrected curves of the map. G. Alignment of hNET between states of apo (cyan) and MIBG-binding (light blue), RMSD = 0.963 Å. EXTENDED DATA FIG. 6

COMPARISON OF SUBSTRATE-BOUND STRUCTURES. A. Superposition of structures of DA-bound dmDAT (light orange, PDB ID: 4XP1) and MIBG-bound hNET (light blue). A zoom-in view of the

substrate-binding site is displayed on the right. B. Superposition of structures of NE-bound dmDATNET (light cyan, PDB ID: 6M2R) and MIBG-bound hNET (light blue). A zoom-in view of the

substrate-binding site is displayed on the right. C. Superposition of structures of GABA-bound (rat) rGAT1 (grey, PDB ID: 8GNK) and MIBG-bound hNET (light blue). A zoom-in view of the

substrate-binding site is displayed on the right. A bean shaped architecture is shown in rGAT1 with GABA fitting into subsite A and subsite C’. EXTENDED DATA FIG. 7 CRYO-EM IMAGE ANALYSIS OF

HNETCRYO-EM BINDING WITH RADAFAXINE. A. Representative raw micrograph (4,043 in total) of hNETcryo-EM binding with radafaxine. B. Representative 2D class averages of hNETcryo-EM binding

with radafaxine. C. The cryo-EM data processing workflow for particle picking, 2D classification, and 3D classification to reach a final three-dimensional reconstruction of hNETcryo-EM

binding with radafaxine at 3.0 Å. D. Angular distribution of all particle images that contributed to the final 3D map, calculated in cryoSPARC. E. Fourier shell correlation (FSC), indicating

the resolution at the 0.143 threshold of unmasked, loosen, tighten, corrected curves of the map. F. Local resolution distribution of the map of hNETcryo-EM binding with radafaxine. G.

Comparing the structures of hNET between states of apo (light cyan) and radafaxine-binding (light pink), RMSD = 1.138 Å. H. RMSD change of the radafaxine pose with NET protein in 100 ns MD

simulation (n = 3). For description of the simulation system, see Supplementary Tables 1 and 2. EXTENDED DATA FIG. 8 COMPARISON OF RADAFAXINE-BOUND HNET STRUCTURE WITH INHIBITOR-BOUND

INWARD-FACING STRUCTURES OF OTHER TRANSPORTERS. A. Un-normalized uptake assay of hNET mutations related to the radafaxine and MIBG-binding sites in Fig. 4f. Data are mean ± s.d. (Empty

vector group n = 25 and others are consistent with Fig. 4f). B. Surface labelling of mutations related to the radafaxine and MIBG-binding sites in Fig. 4f. Data are mean ± s.d. (Empty vector

n = 17; hNETwt n = 18; S318A, D75A, G149Y, Y152A, F317A, and F323Y n = 6; V148A and F323A n = 7; M424A n = 8). C. Superposition of ibogaine-bound hSERT (orange, PDB ID: 6DZZ) and

radafaxine-bound hNET (light pink). The substrate-binding site enclosed by TMs 1, 3, 6, 8 is for structural alignment (RMSD 1.842 Å). Ibogaine and its interacting residues appear as orange

sticks, while Radafaxine in magenta. The inhibitor-binding site is zoomed on the right. D. Superposition of tiagabine-bound hGAT1 (yellow, PDB ID: 7SK2) and radafaxine-bound hNET (light

pink). The substrate-binding site enclosed by TMs 1, 3, 6, 8 is for structural alignment (RMSD 1.818 Å). Tiagabine and its interacting residues are coloured yellow, while hydrogen-bonds as

grey dashed lines. Radafaxine appears in magenta, with the inhibitor-binding site zoomed on the right. E. Superposition of cmpd1-bound hGlyT1 (pale green, PDB ID: 6ZBV) and radafaxine-bound

hNET (light pink). The substrate-binding site enclosed by TMs 1, 3, 6, 8 is for structural alignment (RMSD 1.323 Å). Cmpd1 and its interacting residues appear as green sticks, while

hydrogen-bonds in grey dashed lines. Radafaxine appears in magenta, with inhibitor-binding site zoomed on the right. Source Data EXTENDED DATA FIG. 9 COMPARISON OF HNET STRUCTURE WITH THE

STRUCTURES OF OTHER TRANSPORTERS. A. Structural comparisons of the substrate binding site between the GABA-occluded hGAT1 (lime, PDB ID: 7Y7W) and radafaxine-bound hNET (light pink). The

substrate binding site enclosed by TMs 1, 3, 6, 8 is for structural alignment (RMSD 1.776 Å). B. Close-up of the radafaxine-binding site in A. The inward movement of TM1a toward radafaxine

in hGAT1 (inward-facing conformation) appears an arrow. The movement of Y60 at TM1a of the occluded hGAT1 (F72 in hNET) seems compatible with the binding of radafaxine. C. Structural

comparisons of the substrate binding site between the substrate-free dmDAT (light cyan, PDB ID: 6M0F) and radafaxine-bound hNET (light pink). The substrate binding site enclosed by TMs 1, 3,

6, 8 is for structural alignment (RMSD 3.058 Å). D. Close-up of the radafaxine-binding site in C. The inward movements of TM1a and TM8 toward radafaxine in dmDAT appear as arrows. Movement

of F43 of the outward-facing dmDAT (F72 in hNET) has a steric clash with radafaxine. E. Structural comparisons of the substrate-binding site between the substrate-free dmDAT (light cyan, PDB

ID: 6M0F) and MIBG-bound hNET (light blue). The substrate binding site enclosed by TMs 1, 3, 6, 8 is for structural alignment (RMSD 3.013 Å). F. Close-up of the MIBG-binding site in E. The

inward movements of TM1a and TM8 toward radafaxine in dmDAT appear as arrows. Movement of F43 of the outward-facing dmDAT (F72 in hNET) at TM1a seems compatible with the binding of MIBG. G.

Inhibition mechanism of hNET by radafaxine. The substrate translocation is presumably due to movement of transmembrane helices (TM1 and TM6). Radafaxine is unfavourable to the transition of

hNET from the inward-facing conformation to the outward-facing conformation for inhibition. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Fig. 1: uncropped gels for

Extended Data Fig. 1e,f,l. The cropped regions are indicated by red dashed lines. Supplementary Table 1: reliability and reproducibility checklist for molecular dynamics simulations.

Supplementary Table 2: description of the simulated system. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 1 SOURCE DATA FIG. 2 SOURCE DATA FIG. 3 SOURCE DATA FIG. 4 SOURCE DATA EXTENDED

DATA FIG. 1 SOURCE DATA EXTENDED DATA FIG. 3 SOURCE DATA EXTENDED DATA FIG. 8 RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights

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terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ji, W., Miao, A., Liang, K. _et al._ Substrate binding and inhibition

mechanism of norepinephrine transporter. _Nature_ 633, 473–479 (2024). https://doi.org/10.1038/s41586-024-07810-5 Download citation * Received: 06 December 2023 * Accepted: 10 July 2024 *

Published: 14 August 2024 * Issue Date: 12 September 2024 * DOI: https://doi.org/10.1038/s41586-024-07810-5 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

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