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.