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Supplementary Information Zhou et al. Fig. 1. a. Overall structure of the LeuT-sertraline complex viewed from within the membrane plane. The SSRI molecule binds LeuT between the tips of the EL4 hairpin loop and the extracellular gate formed by residues Arg30, Tyr108, Phe253 and Asp404. Both the R- and S-fluoxetines bind at the same position, as do the TCA molecules. The substrate leucine is colored red, the gate residues purple, and sertraline grey. b. Spatial relationship between the drug-binding site and the substrate-binding site. The sertralinebinding site and the leucine-binding site share four common amino acid residues, Leu25, G26, Y108 and F253, even though the two binding sites do not share common physical space. Dashed lines represent hydrogen bonds. Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602 Fig. 2. Surface representation of the halogen-binding pocket in a, LeuT-sertraline complex and, b, LeuT-R-fluoxetine complex. Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602 Fig. 3. Superposition and comparison of three SSRI structures in their LeuTbound form and free form, the latter previously determined using small molecule crystallography 1,2. a, Sertraline, b, R-fluoxetine and, c, S-fluoxetine. For each SSRI, the main change is a rotation round the bond that connects the halogen-substituted phenyl ring and the rest moiety of the molecule. The torsion angle changes by 180˚ ( of C5-C4-C13-C14), 46˚ (of C3-O5-C6-C7) and 19˚ (of C3-O5-C6-C7), for sertraline, Rfluoxetine and S-fluoxetine, respectively. The free forms are colored in grey, and LeuTbound forms are colored yellow, orange and green, respectively. Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602 Fig. 4. Superposition of LeuT structures when complexed with sertraline, Rfluoxetine and S-fluoxetine, respectively. The drug molecules are removed for clarity. The protein is colored yellow when bound to sertraline, orange when bound to Rfluoxetine, and green when bound to S-fluoxetine. Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602 Fig. 5. Curves showing affinities for antidepressant of human SERT, NET and DAT as measured using binding assays in HEK293 cells, when a residue at their HBP is mutated. The appropriate wild-type transporter was compared. a - c. Binding of [3H]citalopram to the SERT-Ile179Asp mutant in the presence of sertraline, chlomipramine and desipramine. d - f. Binding of [3H]CFT to the NET-Ala77Gly mutant and to the DAT-Ala81Gly mutant (g - i) both in the presence of sertraline, R-fluoxetine and S-fluoxetine. Data points are expressed as percent of specific binding in the absence of antidepressant, and are the mean + S.E.M. of three to five experiments. Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602 Fig. 6. Superposition and comparison of SSRIs and TCAs bound to LeuT. a, Superposition of structures of LeuT-S-fluoxetine and LeuT-desipramine. The amine groups from both S-fluoxetine (SFXT) and desipramine (DSM) are located at the same position, and their amine nitrogen atoms interact with Asp401. The S-fluoxetine molecule is colored green, and the desipramine molecule grey. b, Superposition of structures of LeuT-S-fluoxetine and LeuT-clomipramine. The chloride atom from clomipramine (CMI) does not insert into the same halogen-binding pocket as the SSRIs do. The S-fluoxetine molecule is colored green, and the chloripramine molecule purple Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602 Fig. 7. Sequence alignment between NSS proteins at the EL4 hairpin loop. The arrow indicates an alanine insertion in the SERT sequence. Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602 Table 1. Affinity measurements showing the effect of mutations in the halogen-binding pocket on inhibition of [3H]radioligand binding to SERT-, NET- and DAT-expressing HEK293 cells [3H]Citalopram binding to SERT constructs IC50 (nM) Sertraline Clomipramine Desipramine SERT-WT 4.22±1.37 7.01±1.44 25.0±6.0 SERT-Ile179Asp 290±68* 7.10±1.18 15.4±4.0 [3H]CFT binding to NET constructs IC50 (nM) Sertraline R-fluoxetine S-fluoxetine NET-WT 474±60 193±18 363±43 NET-Ala77Gly 262±17** 99.6±14.8* 159±13** [3H]CFT binding to DAT constructs IC50 (nM) Sertraline R-fluoxetine S-fluoxetine DAT-WT 156±34 396±33 1653±331 DAT-Ala81Gly 41.1±7.3* 170±31* 573±63* Notes: Values are the mean ± S.E.M. of three to five experiments. The significance of measurements is indicated by * (P < 0.05) and ** (P < 0.005) compared with wild-type (Student’s t-test). Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602 References: 1. 2. Caruso, F., Besmer, A. & Rossi, M. The absolute configuration of sertraline (Zoloft) hydrochloride. Acta Cryst. C55, 1712-1714 (1999). Robertson, D.W., Jones, N.D., Swartzendruber, J.K., Yang, K.S. & Wong, D.T. Molecular structure of fluoxetine hydrochloride, a highly selective serotoninuptake inhibitor. J Med Chem 31, 185-9 (1988). Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602