SUPPLEMENTARY INFORMATION

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1 doi: /nature0608 a c pmol L-[ H]Leu / mg LeuT pmol L-[ H]Leu / min / mg LeuT N Cl CMI IMI DMI H C CH N N H C CH N Time (min) N H C nm L-[ H]Leu N H SUPPLEMENTARY INFORMATION b % specific L-[ H]Leu bound d pmol [ H]substrate / mg LeuT No Inhib L-alanine L-leucine L-tryptophan L-tyrosine desipramine 1 mm compound imipramine clomipramine Time (Min) e % specific L-[ H]substrate uptake log [ clomipramine] µm f pmol L-[ H]Ala / min / mg LeuT L-[ H]Ala (nm) Supplementary Figure 1: Functional analysis of LeuT. a, Time course of [ H]Leu transport in the presence of 0 mm TCA (open circles), 1 mm desipramine (DMI) (filled circles), 1 mm imipramine (IMI) (open squares), and 1 mm clomipramine (CMI) (filled triangles). b, Displacement of [ H]Leu binding to LeuT by 1 mm of selected amino acids and TCAs. c, Steady-state kinetics of CMI inhibition of [ H]Leu transport (Michaelis-Menten plot). The open circles, filled circles, and open squares represent data for 0, 200, and 800 µm CMI, respectively. d, Time course of [ H]Leu binding (filled triangles) and transport (open circles) as well as [ H]Ala binding (open squares) and transport (filled circles). e, Dose response curve for CMI inhibition of [ H]Ala (filled circles) and [ H]Leu (open circles) transport. f, Steady-state kinetics of CMI inhibition of [ H]Ala transport (Michaelis-Menten plot). The open circles, filled circles, open squares, and filled triangles represent data for 0, 1, 20, and 50 µm CMI, respectively. Error bars represent the standard error of the mean (S.E.M.) of duplicate (panels b,d,e) or triplicate (panels a,c,f) measurements.

2 a imipramine imipramine 4 EL2 8 1b EL 4 EL2 1b 8 EL a a IL1 IL5 IL1 IL5 L-leucine L-leucine b desipramine desipramine 4 EL2 8 1b EL 4 EL2 8 1b EL a a 2 IL1 IL5 IL1 IL5 L-leucine L-leucine Supplementary Figure 2: Imipramine and desipramine also bind in the putative permeation pathway of LeuT. a, Stereo view of LeuT tilted approximately 15 o from the membrane plane to illustrate the binding sites of imipramine and leucine, both depicted in CPK. Helices whose residues interact with imipramine are coloured. b, Same perspective as panel a, except with the desipramine complex. 2

3 9 EL2 9 EL2 EL EL 8 1b 1b 6a 4 6a a 6b 6b IL1 IL a IL5 CMI 2 IL5 Na1 Na1 CMI 2 Supplementary Figure : Second TCA binding site. Stereo view of LeuT rotated 90 o clockwise from the view in Fig 2c, depicting the position of the second TCA molecule between the 'V' formed by TM4-5 (cyan). For brevity, only the LeuT-leucine-sodium-CMI complex is illustrated, although this second site is present in all four crystal structures. There are no discernible structural alterations between the original (PDB code 2A65) and the LeuT-TCA complexes in this region. Furthermore, the refined temperature factors for the TCAs in this second site are about 2-fold greater than those for the TCAs in the primary, extracellular-facing vestibule site (6-85 versus Å 2 ), suggesting that this second site is not fully occupied and/or is significantly disordered. These observations, combined with the fact that we employed high TCA concentrations (10 mm) in the crystallographic studies, intimate that this second site is probably not relevant to the mechanism by which the TCAs inhibit substrate transport in LeuT.

4 a b A19 (IMI) A19 (DMI) A19 A19 IMI TM1b DMI TM1b D404 HO 2 HO 2 R0 HO (IMI) 2 D404 H 2 O R0 H 2 O HO (DMI) 2 TM10 H O (IMI) 2 TM10 H O (DMI) 2 Supplementary Figure 4: Tip of adopts multiple conformations. Overlay of the TCA binding site in the LeuT-IMI (panel a) and LeuT-DMI (panel b) complexes, illustrating the movement 'up' of residues comprising the tip of (Ala1, Gly18, Ala19). Same colouring scheme as used in Figure d. 4

5 Supplementary Table 1 Kinetic and Dissociation Constants a Dose Response IC 50 of CMI Inhibition of L-[ H]Leu Transport (µm) IC 50 of IMI Inhibition of L-[ H]Leu Transport (µm) IC 50 of CMI Inhibition of L-[ H]Ala Transport (µm) Kinetic Parameters for CMI Inhibition of L-[ H]Ala Transport [CMI] µm K m (nm) V max (pmol/min/mg) Dissociation Rate Constants b 0 mm CMI mm CMI k off (hr -1 ) k off1 (hr -1 ) c ( %) k off2 (hr -1 ) c ( %) a The errors represent the standard error of the mean from duplicate experiments. b Data were fit to either a single or double exponential decay equation, with results from the statistically better fit given in the table. c Number in parentheses represents the percent component of the total observed dissociation. 5

6 Supplementary Table 2 LeuT/TCA Data Collection and Refinement Statistics Clomipramine Imipramine Desipramine CMI/L-Ala Data Collection Space Group C2 C2 C2 C2 Cell Dimensions a, b, c (Å) 88.1, 86.4, , 86.6, , 86.8, , 86.0, 81. α, β, γ (º) 90, 95., 90 90, 95.8, 90 90, 96.9, 90 90, 95.9, 90 Resolution (Å) R merge (%) a 5.6 (52.0) 6.5 (50.) 5.9 (5.1) 4.5 (2.) I/σI a 1.0 (1.) 1.6 (1.9) 19.5 (1.5) 4.6 (2.) Completeness (%) a 89. (51.8) 95.9 (.0) 94. (4.) 90.6 (54.2) Redundancy a.4 (2.2).5 (2.5).6 (2.6) 6.9 (4.4) Refinement Resolution (Å) Unique reflections 45,959 6,941 45,802 4,14 R work /R free (%) 19.8/ / / /21.0 No. atoms Protein Substrate TCA β-og Sodium Water B-factors Overall Protein Substrate TCA TCA β-og Sodium Water RMSD Bond lengths (Å) Bond angles (º) a Number in parentheses represents the highest resolution bin, Å, Å, Å, and Å for the CMI, IMI, DMI, and CMI/L-Ala data sets, respectively. 6

7 Supplementary Methods Before each flux assay described below, proteoliposomes were subjected to two additional freeze/thaw cycles and loaded with fresh buffer I (20 mm HEPES-Tris [ph.0], 100 mm potassium gluconate), a process that minimized the amount of sodium inside the lipid vesicle to support substrate efflux. All functional data were analyzed using GraphPad Prism 4. Inhibition of L-[ H]leu Transport Screen Transport was initiated by diluting LeuT proteoliposomes (0.5 µg LeuT per assay) 200-fold into buffer II (20 mm HEPES-Tris [ph.0], 100 mm NaCl) containing 50 nm [ H]Leu (11 Ci/mmol) and 1 mm NSS substrate or inhibitor (buffer alone for the positive control) at 20ºC. Prior to initiating the reactions, the proteoliposomes had been pre-incubated with either 0 or 1 mm inhibitor for 5 min on ice. Transport activity maintained linearity for up to seven min under these conditions, so the reactions were quenched after six min by diluting 10-fold into ice-cold buffer I and then filtering through Millipore GSWP nitrocellulose filters pre-equilibrated with 2 ml ice-cold buffer I. The filters were subsequently washed three times with 2 ml ice-cold buffer I, placed in 6 ml UltimaGold scintillation fluid, and counted after 5 hours. Uptake into liposomes devoid of protein and subjected to the same experimental conditions was subtracted from the corresponding LeuT data points to determine specific uptake. The entire experiment was performed twice, each time in triplicate, and the data were normalized to that measured in the absence of inhibitor. Inhibition of L-[ H]Leu Binding Screen For these assays, LeuT was expressed and purified as described through the NiNTA step 16 except for the addition of 100 mm alanine in all buffers. The gel filtration chromatography step employed buffer III (HEPES-Tris [ph ], 100 mm choline chloride)

8 containing 20 mm alanine & 1 mm dodecylmaltoside (DDM). Following purification, LeuT was dialyzed extensively against this buffer until the calculated alanine concentration fell below 100 nm. Binding was initiated by adding LeuT to a final concentration of 100 nm in 500 µl buffer II containing 1 mm DDM, 50 nm L-Leu (1:9 H: 1 H; 11. Ci/mmmol), and 1 mm inhibitor (buffer alone for the positive control). Reactions were rotated at room temperature for 2 hours and then terminated by filtering through GSWP nitrocellulose membranes that had been pre-equilibrated with ice-cold buffer II. Filters were subsequently washed three times with 2 ml ice-cold buffer II, placed in 6 ml UltimaGold scintillation fluid, and counted after 5 hours. Nonspecific binding obtained in the presence of 1 mm alanine was subtracted from each data point. The entire experiment was performed twice, each time in duplicate, and the data were normalized to that measured in the absence of inhibitor. Note that polyethyleneimine-treated glass fiber filters (GF/B) were also tried to separate bound from free [ H]Leu, but more reproducible results were obtained with nitrocellulose membranes. TCA Time Course Experiments were conducted as outlined for the transport inhibition screen except that LeuT proteoliposomes were pre-incubated on ice for 0 min with 1 mm TCA, and reactions were quenched at varying time points (0.5, 1, 2, 4, 8, min). The experiment was performed twice, each time in triplicate, and the data were fit to a single exponential. Binding versus Transport Time Course Experiments were conducted as outlined for the TCA time course with three exceptions. First, the reaction buffer contained either 65 nm [ H]Leu (11 Ci/mmol) or 250 nm [ H]Ala (1. Ci/mmol), concentrations which are ~ 40% of the respective Michaelis constants. Second, two sets of LeuT liposomes were prepared, one loaded with 8

9 buffer I to measure transport, the other loaded with buffer II to measure binding. Third, neither the reaction buffer nor the proteoliposomes contained any TCA. The assay was performed twice, each time in duplicate. Dose-Response These experiments were performed as described for the TCA time course except that a single time point of 6 min was used and small aliquots of LeuT proteoliposomes were preincubated on ice for 0 min with varying concentrations of either clomipramine (CMI) (10, 1.6, 100, 18, 16, 562, 1000, 160, µm) or imipramine (IMI) (10, 1.6, 100, 16, 1000, 180, 160, 5620, 10,000, 1,600, 100,000 µm). For inhibition of [ H]Ala transport, the following concentrations of CMI were used: 0.01, 0.016, 0.1, 0.16, 1,.16, 10, 1.6, 100, 16 µm. The external solution (20 mm HEPES-Tris [ph.0], 100 mm NaCl) also contained these same TCA concentrations and either 50 nm [ H]Leu (11 Ci/mmol) or 250 nm [ H]Ala (1. Ci/mmol). Experiments comparing the effect of IMI and CMI on [ H]Leu transport were performed twice, each time in triplicate, while experiments comparing the inhibitory potency of CMI on [ H]Leu vs. [ H]Ala transport were conducted in duplicate. Data were normalized to that measured in the absence of inhibitor and then fit to a sigmoidal dose response equation. LeuT Inhibition Kinetics This experiment was conducted as outlined for the dose response experiments except that the proteoliposomes were not pre-incubated with CMI and the reaction buffer contained varying concentrations of [ H]Leu (11 Ci/mmol) (5, 10, 25, 50, 100, 200, 400 nm) and included either 0, 200, or 800 µm CMI. Experiments examining inhibition of L-[ H]Ala transport included varying concentrations of alanine (1:4 H: 1 H; 14. Ci/mmol) (25, 50, 100, 250, 500, 50, 1000, 2000, 4000 nm) and 0, 1, 20, or 50 µm CMI. Each assay contained 0.05 µg LeuT, and preliminary experiments established that transport activity remained linear 9

10 for up to fifteen minutes under these conditions. Each assay was performed in triplicate, with the entire experiment being replicated twice, and the data were fit to the Michaelis-Menten equation. Dissociation Kinetics LeuT was purified as described for the binding experiments, and binding was conducted as outlined in that section except that a final concentration of 200 nm LeuT was added to 4 ml buffer II containing 1 mm DDM and 200 nm [ H]Leu (11 Ci/mmol). The reaction was rotated as described above and then split into four 900-µl aliquots. Two were diluted 15-fold into buffer containing 100 mm HEPES-Tris (ph.0), 85.5 mm cholinecl, 14.5 mm NaCl, 10 mm cold L-leu, and 1 mm DDM so that the final Na + concentration after dilution was 20 mm. The other two were diluted into the same buffer containing mm CMI. At the indicated time points, 500-µl aliquots were removed from the dissociation reactions and terminated by adding to 500-µl ice-cold buffer containing 20 mm HEPES-Tris [ph.0], 200 mm NaCl. The quenched reactions were then filtered through GSWP nitrocellulose membranes pre-equilibrated with buffer II and treated as outlined above. The entire experiment was performed twice, and error bars represent the standard error of duplicate measurements. Data were fit to either single or double exponential decay equations, with the 0 mm CMI and mm CMI reactions using data up to 60 and 1500 min in the fitting, respectively. Statistical comparisons were made with one-way analysis of the variance (ANOVA) calculations, as implemented in Graphpad PRISM 4.0, with the significance level of the P value set to Structure Determination Diffraction data were collected at 110K at NSLS beamlines X26C (20-sec exposures) and X29A (5-sec exposures) or ALS beamline (-sec exposures) at an X-ray wavelength of 1.1 or 1.0 Å in a 180º or 60 sweep with 1.0º oscillation 10

11 and processed with HKL All four inhibitor complex crystals diffracted to beyond 2.0Å resolution and were indexed in the space group C2. Unit cell dimensions varied slightly from crystal to crystal, but all were approximately a= Å, b= Å, c= Å, β= º. Phases for the CMI complex were obtained via difference Fourier techniques employing the original LeuT structure (PDB ID 2A65) 16 as the starting model. Subsequent model building was accomplished in O 2 with the assistance of sigma-weighted 2Fo-Fc and Fo-Fc maps as well as simulated-annealing Fo-Fc omit maps. Refinement was performed with CNS. This sequence was repeated reiteratively until the R factor and R free values converged, at which point L-leucine or L-alanine, two sodium ions, n-octylβ-d-glucopyranoside, CMI 4, and water molecules were added. Refinement then progressed until R factor and R free converged again. Once the LeuT-leucine-CMI structure was completed, it was subsequently used to procure phases for the LeuTalanine-CMI and LeuT-leucine-IMI 5 complexes, the latter of which was then employed for the desipramine complex. For all four structures, Ramachandran geometry is excellent, with greater than 9% of the residues in the most favored regions and none in disallowed regions. For each data set, the test reflections (4.9% of the total) were selected so that they coincided with those employed in the original structure determination (PDB ID 2A65) 16, which were chosen randomly. The Poisson-Boltzman equation for electrostatic calculations was solved according to the algorithm implemented in the APBS module 6, of PyMOL, and all structure figures were generated with PyMol

12 Supplementary Notes (Literature Cited) 1. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 26, 0-26 (199). 2. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 4, (1991).. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, (1998). 4. Post, M. L. & Horn, A. S. The crystal and molecular structure of the tricyclic antidepressant chlorimipramine hydrochloride: -Chloro-5-(- dimethylaminopropyl)-10,11-dihydro-5h-dibenz[b,f]azepine hydrochloride. Acta Crystallogr. B, (19). 5. Post, M. L., Kennard, O., & Horn, A.S. The Tricyclic antidepressants: imipramine hydrochloride. The Crystal and molecular structure of 5-(- dimethylaminopropyl)-10,11-dihydro-5h-dibenz[b,f]azepine hydrochloride. Acta Crystallogr. B 1, (195). 6. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, (2001).. Lerner, M. G. & Carlson., H. A. (University of Michigan, Ann Arbor, 2006). 8. DeLano, W. L. (DeLano Scientific, San Carlos, CA, 2002).