Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site*

Background: Cocaine interaction with DAT was assessed using the irreversible binding cocaine analog RTI 82. Results: Molecular modeling and peptide mapping identify adduction of RTI 82 to Phe-319 and Phe-320 of rat DAT and human DAT, respectively. Conclusion: Tropane-based pharmacophores bind to DAT in the central substrate site. Significance: Mapping the cocaine-binding site reveals new insights for medication discovery. The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester ([125I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [125I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3␤-(p-chlorophenyl)tropane-2␤-carboxylic acid, 4-azido-3-iodophenylethyl ester ([ 125 I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [ 125 I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies over-

comes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.
The dopamine transporter (DAT) 4 is a presynaptic plasma membrane protein that is responsible for driving the reuptake of dopamine (DA) from the synapse following its vesicular release. This activity controls the magnitude and duration of dopaminergic neurotransmission by maintaining synaptic DA homeostasis (1)(2)(3) and is critical for proper functioning of the nervous system (4). Dysfunction of DAT is hypothesized to contribute to dopaminergic disorders such as schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, autism spectrum disorder, Tourette syndrome, Parkinson disease, and hereditary DAT deficiency syndrome (2,3,(5)(6)(7)(8). Many drugs such as bupropion and methylphenidate that bind to DAT and inhibit transport are used therapeutically to regulate dopaminergic signaling in disease states, whereas others such as cocaine induce psychomotor stimulation and addiction. Many but not all uptake blockers are reinforcing, and some such as the benztropines can counteract a number of cocaine's behavioral effects (9 -11). This suggests the potential for development of improved pharmacotherapies for drug abuse and other DA disorders, but despite years of research, the molecular basis by which structurally distinct inhibitors interact with DAT and induce differential neurochemical and behavioral outcomes remains incompletely understood.
DAT and the related cocaine-sensitive norepinephrine and serotonin transporters (NET and SERT) belong to the neurotransmitter sodium symporter (NSS) subfamily of solute carrier 6 transporters (SLC6A) that share a common topology of 12 transmembrane (TM) domains connected by extracellular (EL) and intracellular (IL) loops (6,12,13). Solute translocation is driven by a Na ϩ -and Cl Ϫ -dependent alternating access mechanism in which the proteins cycle through outwardly and inwardly facing conformations that bind and release substrates on opposite sides of the membrane (14). Major insights into structural mechanisms of NSS proteins have come from the homologous prokaryotic leucine transporter (LeuT) from Aquifex aeolicus, which has been crystallized in conformations corresponding to outwardly facing, occluded, inwardly facing, and inhibitor-bound forms (15)(16)(17)(18)(19)(20). These structures reveal that outwardly facing transporters possess an aqueously accessible external vestibule that opens to a compact central/primary substrate-binding site (S1) composed of residues from TM domains 1, 3, 6, and 8 (15). Binding of substrate, two Na ϩ (Na1 and Na2) ions, and one Cl Ϫ ion to their respective sites triggers conformational changes leading to closure of an extracellular gate that occludes S1, followed by opening of the intracellular gate and generation of an inwardly facing form that releases solutes to the cytoplasm (14,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The S1 site is highly conserved in mammalian transporters, and mutagenesis of many DAT residues near this site, including Phe-76, Asp-79, Val-152, Phe-155, Tyr-156, Asn-157, Phe-319, Val-327, and Ser-421, reduces dopamine transport (31)(32)(33)(34)(35)(36)(37)(38), supporting the participation of these amino acids in substrate recognition or translocation. In particular, Asp-79 in TM1 of DAT and the homologous Asp residues in NET and SERT coordinate the positive charge on monoamine substrates and play an essential role in transport (6,12,13,15,39,40). However, a second, highly controversial substrate site (S2) proposed to play a role in the initiation of transport has been identified in LeuT in the extracellular vestibule above the outer gate (27,(41)(42)(43). The S2 site may also be conserved in mammalian NSS transporters, but its existence and role in NSS function remain a topic of debate.
Intensive efforts to understand the basis for cocaine interaction with DAT, NET, and SERT have relied on site-directed mutagenesis (SDM), substituted cysteine accessibility method (SCAM) (44 -47), and quantitative structure activity relationship (QSAR) of ligand pharmacophores (48,49). Many studies support the formation of a salt bridge between the conserved Asp residue in the unwound region of TM1 and the positively charged tropane nitrogen of cocaine, suggesting that cocaine binds near S1 and competes with substrate for interaction at this crucial site (31,37,38,44,47,50). Numerous other residues, including Leu-104, Phe-105, Ala-109, Asn-157, Tyr-251, Tyr-273, Thr-315, Ser-320, Thr-455, and Ser-459 found throughout core TM domains of DAT, have also been implicated in the binding of cocaine and its analogs (37,(51)(52)(53)(54), but it has been difficult to unambiguously assign antagonist binding functionalities based on mutation-based strategies due to potential indirect effects on protein structure. Overlap of high affinity inhibitor binding with the S1 site is supported by biochemical studies (38,44,47,55) and by recent structures of Drosophila (d) DAT and a LeuT/SERT hybrid co-crystallized with antidepressants (40,56). However, some DA transport inhibitors lack the charged nitrogen necessary to form the salt bridge with Asp-79 (57)(58)(59), suggesting that inhibitor binding can occur without this interaction and thus may not be limited to the S1 pocket (60). This possibility is supported by crystal structures of LeuT complexed at relatively low affinity with several selective serotonin uptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs) in regions that overlap with S2 (16 -18, 56). However, as yet no crystal structures of NSS transporters complexed with cocaine have been obtained to distinguish between these possibilities, and many unknowns remain regarding the mechanistic relationship of S1 and S2 sites to each other and to the actions of cocaine and other categories of transport inhibitors.
In this study, we investigated the site of cocaine binding on DAT by combining the strategies of comparative modeling, small molecule docking, molecular dynamics, peptide mapping, and SCAM protection to identify the attachment site for the irreversible cocaine analog [ 125 I]3␤-(p-chlorophenyl)tropane-2␤-carboxylic acid, 4Ј-azido-3Ј-iodophenylethyl ester ([ 125 I]RTI 82) ( Fig. 1) (50). RTI 82, cocaine, and the commonly used cocaine analog (Ϫ)-2␤-carbomethoxy-3␤-(4-fluorophenyl)tropane (CFT) share a common pharmacophore consisting of the tropane ring and 3␤-phenyl group that direct high affinity binding (48,49). All of these compounds bind noncovalently to DAT, but [ 125 I]RTI 82 contains a photoactivatable 4Ј-azido-3Јiodophenylethyl ester (AIP) moiety that forms a highly reactive singlet nitrene that reacts with C-H or N-H groups when the sample is irradiated with ultraviolet light, thus forming a covalent bond with the protein (61,62). Using irreversible labeling as a strategy for determining the site of ligand-protein interaction, we previously demonstrated that [ 125 I]RTI 82 cross-links to human (h) DAT within a 40-residue region encompassing TM6 (63). Utilizing this information as a molecular constraint in flexible docking of RTI 82 to rat (r) DAT homology models based on "outward-occluded" and "open-to-out" LeuT crystal structures (Protein Data Bank codes 2A65 and 3F3A), we identify RTI 82 binding in the S1 pocket in an orientation that suggests TM6 residue Phe-319 as the likely site of AIP adduction. This prediction was verified by focused mapping of the [ 125 I]RTI 82 attachment site using methionine substitution mutagenesis and cyanogen bromide (CNBr) digestion and by SCAM analyses that indicate protection of S1 but not S2 residues by cocaine and RTI 82. These positive function findings physically localize the tropane pharmacophore to the S1 site in transport-competent mammalian transporters and provide information crucial for refinement and experimental verification of results obtained by homology modeling.

EXPERIMENTAL PROCEDURES
Comparative DAT Homology Model Construction-The three-dimensional coordinates from the outward-occluded and open-to-out crystal structures (Protein Data Bank IDs 2A65 and 3F3A, respectively) of the LeuT from A. aeolicus (Uniprot accession number O67854), were mapped onto rDAT sequence based on the comprehensive sequence alignment from Beuming et al. (64) to construct the occluded and outward facing homology models of rDAT. The missing atomic densities in the loop regions of the transporter were rebuilt using the kinematic closure method (65) in Rosetta3.1 (66). The N and C termini, which are missing in LeuT, were truncated in the rDAT model to yield a structure consisting of amino acids 65-601, which includes TMs 1-12. Side chains for all residues in the protein were built using Rosetta's Metropolis Monte Carlo rotamer search algorithm (67). The starting rDAT model was subjected to six iterative rounds of relaxation and minimization using Rosetta3.1 to produce an ensemble of 100 rDAT models each for 2A65 and 3F3A. The top 10 ranked models based on the Rosetta E total score for each template were verified manually and carried forward for docking.
RosettaLigand Docking-The three-dimensional structure for the RTI 82 ligand was built using "builder" of the Molecular Operating Environment (68) program and energy-minimized and was output as a mol file. The mol file was used as input for Rosetta3.1 to generate 1000 RTI 82 rotamers. The ligand parameter file, which assigns Rosetta atom types to the molecule for use in RosettaLigand 3.1 (RL), was generated using the Rosetta3.1 script "molfile_to_params.py". Because of the multiple resonance states of the azide group and lack of an available parameterized force field for this chemical group, we utilized the atom type Nhis which was assigned to the azide nitrogens by Rosetta. Manually changing the atom types to Nhis-Nlys-Nlys, Nhis-NH2O-NH2O, Nlys-Nlys-Nlys, or Ntrp-Ntrp-Ntrp to test other bond hybridization and charge states resulted in markedly poorer docking energy scores. For the ensemble of 10 rDAT models built from the LeuT 2A65 structure, the collection of 1000 RTI 82 conformers was randomly docked into the structures using RL (69). For more information regarding the RL docking protocol, see Combs et al. (70). Briefly, RTI 82 was placed at a coordinate in the rDAT model equivalent to the substrate-binding site in LeuT and allowed to randomly trans-late within a 10-Å sphere. Acceptable ligand translations underwent up to 1000 random rotations to energetically optimize the pose and minimize clashes. Residue side chains within 6 Å of RTI 82 were repacked using a Metropolis Monte Carlo simulated annealing algorithm and scored using the knowledge-based Rosetta energy function interface_delta, which is the difference between the total binding energy E (transporter ϩ ligand) and the sum of the energy of the individual components E (transporter) ϩ E (ligand) separated by 500 Å. A total of 25,000 ligand dockings were conducted for the 10 models from 2A65. The process was repeated for 3F3A-based models.
The resulting rDAT-RTI 82 complexes were ranked based on the interface_delta scores, and the top 10% of each of the 10 rDAT structures was compiled and evaluated by ligand-based r.m.s.d. clustering (70). The top scoring 10% rDAT-RTI 82 complexes were evaluated by two methods as follows: 1) complexes found in the largest r.m.s.d.-based clusters were ranked by score, and 2) rDAT-RTI 82 complexes were filtered for RTI 82 poses with the azido moiety within 5 Å of residues of TM6 and the tropane nitrogen within 5 Å of residue Asp-79.
Induced Fit Docking-The top Rosetta rDAT homology models of the 3F3A and 2A65 templates were modified to include two Na ϩ ions and one Cl Ϫ ion in their reported positions (15,71) using Maestro (Schrödinger, LLC, Portland, OR). The protein was relaxed, optimized for hydroxyl and thiol groups, and minimized using default options in the protein preparation module of the Maestro software package (Maestro Schrödinger, LLC, Portland, OR). RTI 82 conformers were generated using the LIGPREP (Version 2.5) protocol. RTI 82 conformers were docked into the rDAT homology models containing Na ϩ and Cl Ϫ ions utilizing induced fit docking (IFD) protocol (72). As the Na ϩ and Cl Ϫ ions were relaxed during the IFD analysis, we verified that the ions remained coordinated by the same side chains before and after docking. The final ranking of the docked poses comes from the composite score, which is GlideScore ϩ 0.05 ϫ PrimeEnergy.
Molecular Dynamic Simulations-Molecular dynamic simulations (GROMACS) were conducted on RTI 82 docked rDAT homology models with incorporated Na ϩ and Cl Ϫ ions. The top ranked RTI 82 docked rDAT poses were embedded in the center of a pre-equilibrated 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) lipid bilayer (along the z axis, coinciding with the normal of the POPC bilayer) using Visual Molecular Dynamics package (73) The dimensions of the simulation box were 20 ϫ 20 ϫ 14 Å containing one protein, one ligand, ϳ50,000 TIP3P water molecules, and ϳ225 POPC molecules. 186 Na ϩ and 188 Cl Ϫ counter-ions were added to obtain electroneutrality for the system reaching a salt concentration of 150 mM. All simulations were carried out with GROMACS version 4.5.4 (74) using CHARMM27 force field under periodic boundary conditions. SwissParam (75) was used to generate RTI 82 topology and parameter files, based on the Merck molecular force field, that are compatible with CHARMM and GROMACS. Isothermal-isobaric ensemble with velocity scaling (V-rescale) thermostat and Parrinello-Rahman barostat was used to perform simulations. For the CHARMM force field operating in GROMACS, electrostatics were calculated using particle mesh Ewald (76) with appropriate cutoffs as follows: rlist ϭ 1.3; rcoulomb ϭ 1.3; rvdw ϭ 1.2; vdwtype ϭ switch; and rvdw_switch ϭ 0.8. Fourier spacing of 0.12 nm and a particlemesh Ewald electrostatic order of 4 was employed. V-rescale thermostat with a coupling constant of 0.1 ps was used to separately couple protein, lipid, and solvent, including water and ions. The pressure was coupled using the Parrinello-Rahman algorithm at 1 bar with a coupling constant of ϭ 1 ps, using a uniform compressibility of 4.5 ϫ 10 Ϫ5 bar Ϫ1 . The coordinates were saved every 100 ps with an integration time step of 2 fs. The LINear Constraint SolVer (LINCS) algorithm was used to restrain all bond lengths (77). The minimized, ion-bound rDAT-RTI 82 complexes in the POPC bilayer were further equilibrated for 4 ns in GROMACS at a temperature of 303 K, by fixing the position of the docked complex through application of position restraints of 1000 kJ⅐mol Ϫ1 ⅐nm Ϫ2 on each heavy atom, whereas lipids and water were allowed to move normally. After an initial equilibration for 4 ns, the production runs were performed for 60 ns. The pressure was maintained at 1 atm using semi-isotropic pressure coupling to a Parrinello-Rahman barostat with a coupling constant of 5 ps. Conformations resulting from the production phase of each simulation were stored at intervals of 100 ps and analyzed. PyMOL (78) was used to generate the molecular graphic diagrams.
Cell Culture and Site-directed Mutagenesis-For photoaffinity labeling experiments, WT and mutant cDNAs in pcDNA3.1/ His vector were stably expressed in Lewis lung carcinoma-porcine kidney (LLC-PK 1 ) cells. All mutants in the study were generated using the Stratagene QuikChange kit and verified by sequencing (Northwoods DNA, Solway MN, Eurofins MWG Operon, Huntsville, AL). Mutants for SCAM analysis were generated in the E2C background (C90A and C305A) of pcDNA3-rDAT and expressed transiently in HeLa or HEK-GripTite cells (Invitrogen) using Lipofectamine 2000 (Invitrogen) or TransIT-LT1 (Mirus). WT and mutant r/hDAT cells were maintained in a humidified chamber with 5% CO 2 at 37°C in ␣-minimum essential medium (AMEM: 5% fetal bovine serum, 2 mM L-glutamine, 200 g/ml G418, and 100 g/ml penicillin/streptomycin) for LLC-PK 1 cells or Dulbecco's modified Eagle's medium (DMEM: 10% FBS, 100 units/ml penicillin G, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B) for HeLa and GripTite cells (600 g/ml G418). Once plated, the S321M line was grown at 29°C for better expression of mature DAT.
[ 3  Binding was performed in triplicate with nonspecific binding determined with 30 M mazindol. Uptake and binding reactions were quenched by washing cells twice with ice-cold KRH followed by solubilization of cells with radioimmunoprecipitation assay buffer (RIPA: 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 7.5) containing protease inhibitors. Lysates were assessed for radioactivity by liquid scintillation counting and for protein content by the BCA method. DAT expression was assessed by immunoblotting with hDAT monoclonal antibody 369 (mAb 369, Chemicon International) with band densities quantified using Bio-Rad Quality One software. All assays were performed in triplicate and repeated at least three times. Results were normalized for DAT levels and expressed relative to the WT protein set at 100%. Statistical significance was determined by ANOVA with post hoc Dunnett test with significance set at p Ͻ 0.05.

H]Dopamine Uptake and [ 3 H]CFT Binding Assays-WT
RTI 82 Synthesis and Radioiodination-RTI 82 and the amino precursor of [ 125 I]RTI 82 were synthesized using modifications (50) of the original procedure. Radioiodination was conducted as described previously (79).
Photoaffinity Labeling and CNBr Peptide Mapping-These procedures were performed as described previously (63,80). WT and mutant cells were washed twice with ice-cold KRH and incubated with 5 nM [ 125 I]RTI 82 in KRH buffer for 2 h on ice in the presence or absence of 30 M (Ϫ)-cocaine. Cells were irradiated with ultraviolet light (254 nm) for 5 min to covalently attach the ligand to the protein. The cells were washed twice with cold KRH to remove unbound ligand and were lysed with RIPA containing protease inhibitor for 30 min on ice with shaking. The lysates were centrifuged at 20,000 ϫ g for 12 min at 4°C, and supernatants were subjected to electrophoresis on 8% Tris-glycine polyacrylamide gels for gel purification or were immunoprecipitated with anti-His antibody and electrophoresed on 4 -20% Tris-glycine polyacrylamide gels, followed by autoradiography. For CNBr analysis, photolabeled DAT bands were excised from the gel, electroeluted, dialyzed against purified H 2 O, lyophilized to dryness, and suspended in vehicle. Aliquots were counted in a scintillation counter, and equal amounts of radioactivity were subjected to peptide mapping. To assess fragment deglycosylation, samples were treated with vehicle or 150 units of neuraminidase (New England Biolabs) for 1 h at 37°C followed by addition of 3000 units of PNGase F (New England Biolabs) for 15 h at 22°C prior to treatment with CNBr. For CNBr proteolysis, samples were resuspended in 70 l of 70% formic acid with or without addition of 1 M CNBr for 24 h at 22°C in the dark. Reactions were quenched with 1 ml of purified water, and samples were lyophilized to dryness, followed by four additional rounds of resuspension with water and lyophilization. Samples were resuspended in 4SB buffer (50 mM Tris, 5 mM EDTA, 4% SDS, pH 7.4) and subjected to acetone precipitation followed by centrifugation at 20,000 ϫ g for 15 min. Pellets were solubilized in sample loading buffer (0.625 M Tris-HCl, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.005% bromphenol blue) and analyzed by SDS-PAGE/autoradiography on 4 -20% gels. For each experiment, 2-3 mutants were photoaffinity-labeled and analyzed with WT DAT exactly in parallel, and all results were replicated at least three times. CNBr peptide masses were calculated using PeptideCutter via ExPASy.
RTI 82 Potency in Phe-319 Mutants-HeLa cells were plated at a density of 50,000 cells/cm 2 in 24-well culture plates, incubated for 24 h, and transfected with rDAT constructs using GeneCellin (Bulldog Bio) (1 l per 200 ng of DNA). Following transfection (24 h), cells were washed with 37°C KRH buffer and then incubated with 50 nM or 5 M [ 3 H]DA and increasing concentrations of nonradiolabeled RTI 82 without photoactivation. Transport was terminated after 15 min by washing with cold KRH. Cells were then dissolved in MicroScint 20 (PerkinElmer Life Sciences) scintillation fluid, and radioactivity was quantified by a TopCount scintillation counter. Specific uptake was determined by subtracting uptake observed in nontransfected cells. For binding analyses, transiently transfected cells were washed twice with ice-cold KRH buffer followed by addition of various concentrations of competitor and 10 nM [ 3 H]CFT. Samples were incubated at 4°C for 2 h, washed twice with KRH, and dissolved in MicroScint 20 scintillation fluid, and radioactivity was quantified as above. Nonspecific binding was determined with 30 M mazindol. All experiments were performed in triplicate and repeated in three or more separate assays. IC 50 data were analyzed using a one-way ANOVA and post hoc Dunnett test (Prism 4, GraphPad).
SCAM Protection Analysis of S1-and S2-binding Sites-HeLa or GripTite cells were plated and transfected as described above. For cocaine protection, HeLa cells were treated 24 h post-transfection with 5 M (Ϫ)-cocaine or vehicle for 10 min at 37°C followed by addition of 5 mM MTSET for 10 min at room temperature in PBS/CM buffer (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 0.1 mM CaCl 2 , 1.0 mM MgCl 2 , pH 7.4). Post-treatment, cells were washed with PBS/CM and incubated in KRH buffer at 37°C for 30 min to allow for cocaine dissociation. Cells were then washed with KRH and assayed for transport activity by incubation with 50 nM [ 3 H]DA. Uptake was terminated after 15 min by washing with cold KRH. Cells were dissolved in MicroScint Beta (PerkinElmer Life Sciences), and radioactivity was quantified as described above. All experiments were repeated in three or more separate assays. Cocaine inhibition of uptake was verified in samples that did not undergo washout. Data were analyzed using a two-way ANOVA followed by a post hoc Bonferroni test (Prism 4). For RTI 82 protection studies, GripTite cells (Invitrogen) were processed 48 h post-transfection by washing twice with PBS/CM and incubating with 10 or 50 M RTI 82 or vehicle for 5 min followed by addition of 0.1 mM MTSEA-biotin (Biotium). Cells were then processed as described previously (44) to obtain cell lysates that were incubated with NeutrAvidinagarose resin (ThermoFisher) to extract surface proteins labeled by MTSEA-biotin. Equal amounts of protein from total samples as determined by BCA assay (ThermoFisher) and equivalent volumes of surface protein pools were processed by SDS-PAGE and immunoblotting using anti-DAT monoclonal antibody (mAb 16). Surface and total DAT levels were quantitated from the density of immunoblot bands using ImageJ (National Institutes of Health). Surface values were normalized to total DAT levels and are expressed as percent of untreated samples. Data were analyzed using a paired t test (Prism 4).

RESULTS
Computational Docking of RTI 82-To facilitate elucidation of RTI 82 attachment to DAT, we generated LeuT-based comparative models of DAT followed by analysis of RTI 82 binding poses obtained from two independent computational docking methodologies, RL and IFD. For both methods, docking of RTI 82 was performed using rDAT homology models based on the LeuT outward-occluded (2A65) and open-to-out (3F3A) crystal structures. For RL, an ensemble of starting structures was used (10 for each conformation) (81), and for IFD the lowest energy (top-scoring) structure from Rosetta homology modeling was refined using Protein Preparation Wizard (Schrodinger Software Suite), resulting in one structure each from the 2A65and 3F3A-based templates. Best fit ligand-docked poses from these methods (see below) were then analyzed by molecular dynamics.
RosettaLigand-The rDAT-RTI 82 complexes obtained from RL docking, termed "decoys," were ranked according to their interface_delta score that measures the interaction energy between the ligand and the protein (70). The top 10% (n ϭ 2500 for each model) was evaluated by clustering of the ligand poses using CLUSTER with a 5 Å cutoff (70). The analysis resulted in five clusters of which clusters one and four contained the largest number of decoys (517 and 422, respectively) with the ligand occupying the central S1-binding site, and the remaining clusters contained between 11 and 178 decoys. In clusters one and four, the top 50 scoring decoys were initially filtered using molecular distance constraints from biochemical data to identify complexes where the RTI 82 azido group was within 5 Å of any atoms between residues 272-344 of rDAT, and the tropane nitrogen of RTI 82 was positioned within 5 Å of the Asp-79 side chain (31,63).
In the 2A65-based outward-occluded models ( Fig. 2A), the top 10 scoring decoys that satisfied the molecular constraints were chosen for further analysis. Notably, the top scoring models resulting from this selection method were identified as the highest scoring models prior to applying the distance filters, indicating that the computational docking methods were capable of identifying poses that were corroborated by biochemical cross-linking. Additionally, before the filtering step, all of the top-scoring decoys positioned the negatively charged carboxyl oxygen of Asp-79 within 3.5 Å of the positively charged tropane N, supporting salt bridge formation as a major contributor to energetically favorable binding. The top scoring 2A65-based decoys also displayed interaction between the RTI 82 3␤-phenyl chloride group and TM3 residue Asn-157, an interaction that has previously been suggested as important for CFT and benztropine analog binding (37,54). In this "best pose," which fulfilled multiple major biochemical constraints ( Fig. 2A, green  sticks), the AIP moiety was oriented toward the external vestibule with the arylazide positioned proximal to the phenyl side chain of Phe-319, suggesting this residue as the likely site of RTI 82 adduction.
In the 3F3A-based outward-facing models, analysis of the decoys generated from docking of RTI 82 to rDAT revealed that the AIP moiety fell within 4 Å of TM6 in 310 of the top scoring docked complexes. The azido group was positioned proximal to Phe-319 and Phe-325 in 208 and 224 of the complexes, respectively, with many of the poses placing the azido within 4 Å of both residues simultaneously. However, none of the 310 poses satisfied the second distance constraint between the tropane N and the Asp-79 side chain, and therefore, these decoys were not further analyzed by MD. It is possible that the inability of Rosetta to account for Na ϩ and Cl Ϫ during the minimization of the ensemble of DAT structures may have had more of a negative impact on the more "open" 3F3A-based models than the "occluded" 2A65-based models and resulted in structures that were less able to approximate the native, ligand-bound conformation resulting in the inability to obtain a biochemically supported binding pose. However, given that Phe-325 was the most frequently identified candidate residue from these "outwardfacing" models, its potential as a possible site of adduction was investigated in CNBr peptide mapping studies.
Induced Fit Docking-Docking analyses using IFD in the Schrödinger software suite (Fig. 2B) were conducted to allow for the integration of energetic contributions of Na ϩ and Cl Ϫ ions to ligand binding during the docking step (15,39,71,82,83). This alternative approach yielded 12 and 34 decoys from 2A65-and 3F3A-based models, respectively. Although the best scoring decoys consisted of RTI 82 poses in which the tropane N could be either protonated or unprotonated, only poses with protonated ligand were carried forward based on current data (84) and analysis using FIXPKA in QUACPAK Version 1.5.0 (85), which predicts protonation of the group at pH 7.4. In both the 2A65-and 3F3A-based models, the top scoring poses positioned RTI 82 in the S1 pocket such that the AIP moiety was oriented toward Phe-319, with astacking interaction between the phenyl rings of the AIP group and the Phe-319 side chain (Fig. 2B). For the 2A65-based model, the two top scoring poses differed in their proximity to Phe-319 by only 0.011 Å, with the best energy scoring pose being selected (Fig. 2B, yellow  sticks). For the 3F3A-based models, the AIP groups in the top two poses were proximal to the Phe-319 side chain with distances of 3.5 and 3.9 Å and the docking scores of Ϫ11.45 and Ϫ12.12, respectively. As the docking scores were similar, the best 3F3A pose (Fig. 2B, blue sticks) was selected based on proximity of Phe-319 and the azido group of RTI 82.
MD Simulation of RL and IFD Docked RTI 82 Poses-To further refine the RTI 82 pose in the rDAT-RTI 82 complexes, the best-docked structures from RL and IFD analyses were embedded in a POPC lipid bilayer and refined by MD simulation for 60 ns in GROMACS (74). The r.m.s.d. analysis of the ligand atoms and C␣ atoms of DAT during simulation revealed that RTI 82 was stable in the binding pocket throughout the simulation (Fig. 3). In particular, we monitored the stability and time-dependent changes of two key protein-ligand interactions during simulation as follows: 1) the carboxylate oxygen of Asp-79 and the protonated tropane N, and 2) the amide nitrogen of Asn-157 and the 3␤-phenyl chloride. In all simulations, a stable (occupancies of 58% for RL-2A65, 52% for IFD-2A65, and 98% for IFD-3F3A) salt bridge interaction distance (3.5 Å) was observed between the RTI 82 tropane N and the Asp-79 carboxyl oxygen (red and black lines in Fig. 3, A-C). In the RL-2A65 and IFD-3F3A simulations, this interaction is maintained through periodic reciprocal reorientation of the O␦1 and O␦2 atoms. In contrast, using a 4-Å cutoff, the predicted polar interaction between Asn-157 and the RTI 82 3␤-phenyl chlo-  OCTOBER 24, 2014 • VOLUME 289 • NUMBER 43 ride was observed to be stable (70% occupancy) only in the RL-docked pose (Fig. 3A, blue line), whereas negligible interaction (2-4% occupancy) was found in the IFD-docked poses (Fig. 3, B and C, blue lines).

Tropane-binding Site on DAT
The best poses obtained by RL and IFD before and after MD simulation are illustrated in Fig. 4. All of the docking methodologies, irrespective of the input rDAT model, yielded RTI 82 poses with the AIP arm extended out of the S1 pocket and toward the external vestibule (Fig. 4, A-C). In RL (Fig. 4A), RTI 82 docked in the outward-occluded model exhibited a structure with the AIP group in a bent conformation (green sticks), and after MD simulation, the AIP moiety displayed an extended and more linear conformation (Fig. 4A, magenta sticks). This extended AIP configuration was also found in the IFD-generated pose from the occluded form, both prior to (yellow sticks) and after (black sticks) MD simulation (Fig. 4B). This extension may be stabilized by an interaction between the AIP azido moiety and outer gate residue Arg-85 (Fig. 4, A and B). In contrast, in the IFD-generated open-to-out complex (Fig. 4C, blue sticks), the AIP group was slightly bent prior to simulation such that it formed astacking interaction with Phe-319 (blue arrowhead). During simulation of this docked complex, the AIP functional group (Fig. 4C, brown sticks) was repositioned between TM6 and TM10 near Ala-479, yet maintainedstacking with Phe-319 (brown arrow).
Notably, the position of the RTI 82 tropane pharmacophore was relatively unchanged in all poses following simulation, indicating that this functionality is the major contributor to ligandprotein interaction. In fact, during simulation, we consistently observed a slight translation of the tropane ring deeper into the binding pocket (Fig. 4, A-C). This was accompanied by similar adjustments of interacting residues Phe-76, Asp-79, Phe-325, and Val-327, which maintained their coordination with the ligand (Fig. 4, A-C). The interaction of the RTI 82 3␤-phenyl chloride with Asn-157 was also maintained during MD simulation of the RL pose (Fig. 4A) but was lost in both IFD decoys (Fig. 4, B and C). The validity of our poses is further supported by recent crystal structures of the LeuT/SERT hybrid (56) and dDAT (40) transporters that show binding of SSRI and TCA antidepressants to S1. The binding pose of nortriptyline at the dDAT S1 pocket (40) overlaps substantially with our RTI 82 pose as shown in Fig. 4D. These findings indicate that many classes of high affinity neurotransmitter transporter inhibitors may assume similar poses within the S1 pocket.
Analysis of 4Ј-Azido-3Ј-iodophenyl Moiety Interactions with Phe-319 -Our poses showing the proximity of the RTI 82 AIP group to the Phe-319 side chain suggested aromatic stacking as the basis for their interaction. To investigate this possibility, we generated a panel of conservative and nonconservative Phe-319 mutants (F319Y, F319W, F319M, F319C, and F319D)    assess transport, the F319Y, F319W, and F319M mutants showed 8 -43% of the WT DAT activity level, whereas uptake was undetectable in the F319C and F319D mutants ( Table 1). The significant reduction in high affinity transport in F319M, F319C, and F319D DATs is consistent with the loss of aromatic interaction needed for Phe-319 to interact with its gating partner Tyr-156 (12,15,18). RTI 82 transport inhibition potencies did not differ between the WT protein or the Phe-319 mutants substituted with aromatic (Trp or Tyr) or nonaromatic hydrophobic (Met) residues, indicating thatinteractions between the RTI 82 AIP group and the side chain of Phe-319 do not provide major contributions to RTI 82 binding. However, the potency of RTI 82 to inhibit [ 3 H]CFT binding in F319C and F319D DATs was reduced by 4.3-and 7.1-fold (Table 1), indicating that inhibitor effects require hydrophobicity at Phe-319. We also found that when higher concentrations of DA (5 M) were used for uptake, the F319C and F319D mutants remained nonfunctional, suggesting that the charge substitution at this position may impact substrate affinity. However, transport activity could be restored in the F319C mutant to 79% upon application of 3.5 mM DA, whereas the F319D mutant remained nonfunctional.
CNBr Peptide Mapping of [ 125 I]RTI 82-Labeled TM6 Mutants-We then sought to validate the prediction from computational modeling that Phe-319 was the likely site of RTI 82 adduction. For this, we generated methionine (Met) substitutions of residues flanking Phe-320 in hDAT, which corresponds to Phe-319 in rDAT, for use in CNBr peptide mapping. hDAT is identical to rDAT in amino acid sequence across the region mutated for these studies (hDAT residues 318 -330), and it was used because the two additional Met residues present in EL2 and EL3 of rDAT (Fig. 5) would significantly complicate the CNBr digestion analyses. In addition, to further strengthen the interpretation of the proteolysis patterns, we introduced all TM6 Met substitutions in an M272L background to eliminate Met-272 as a CNBr proteolysis site (63). In hDAT, Met-272 is the only Met between TM2 and TM7. Thus, in the M272L background, the introduction of Met into TM6 would result in generation of CNBr fragments of ϳ50 kDa that would extend from TM2 (Met-106, Met-111, or Met-116) to the inserted Met or CNBr fragments of ϳ6 kDa that would extend from the inserted Met to Met-371 in TM7 (Fig. 5). The masses of the labeled CNBr fragments produced thus indicate the relative positions of the inserted Mets and the site of [ 125 I]RTI 82 adduction. For ease of discussion, we refer to the double mutants with TM6 Met substitutions in the M272L background solely by the TM6 mutation (e.g. V318M hDAT indicates M272L/V318M hDAT) and to the CNBr fragments by the flanking Mets, although the N terminus of each fragment is the residue following a proteolyzed Met.
When normalized for transporter expression levels, the mutants showed [ 3 H]DA uptake values that ranged from ϳ20 to 60% of the WT protein, except for F320M and S321M, which exhibited Ͻ10% of the WT activity (Fig. 6B). Loss of transport in these mutants is consistent with their functional roles, as Phe-320 is proposed to act as a substrate pocket gating residue, and Ser-321 coordinates Na ϩ at the Na1 site (15,40). [ 3 H]CFT binding for the mutants was impacted less than transport, ranging from ϳ60 to 90% of WT levels when normalized for expression (Fig. 6B), and all forms showed [ 125 I]RTI 82 photoaffinity labeling that was fully blocked by cocaine (Fig. 6C), indicating that the mutations did not substantially disrupt the cocaine binding pocket.
For peptide mapping studies, the photolabeled proteins were gel-purified and subjected to treatment with vehicle (formic acid) or CNBr, followed by SDS-PAGE/autoradiography. Within each experiment, equal amounts of radioactivity for WT and mutant forms were analyzed to allow for direct comparison of peptide fragment production. Fig. 7A shows a compilation of representative peptide maps produced from three or more independent replicates for each mutant, with schematic diagrams indicating the origin of the labeled fragments in the primary sequence and site of [ 125 ]RTI 82 adduction (star symbol). Full-length, unproteolyzed DAT migrates at ϳ90 kDa (Fig.

Tropane-binding Site on DAT
7A, odd-numbered lanes) with no low molecular weight fragments observed. Aggregates seen at Ն180 kDa are most likely induced by the formic acid treatment, as they were not seen in samples subjected directly to electrophoresis. CNBr treatment of WT hDAT produced a labeled fragment of ϳ11 kDa (Fig. 7A,  lane 2, arrow a), as we demonstrated previously (63), that corresponds to the region between Met-272 and Met-371 (calculated mass 10.6 kDa, shaded region in schematic diagram a), as well as larger fragments that likely arise from missed cleavage of Met-272. CNBr treatment of [ 125 I]RTI 82-labeled V318M and C319M hDATs produced fragments of ϳ6 kDa (Fig. 7A, lanes 4  and 6, arrow b) that are consistent with peptides extending from the inserted Mets to Met-371 (calculated masses ϳ5.6 kDa; shaded region in schematic diagram b), with higher molecular weight fragments in these samples also indicating missed cleavage of the inserted Mets. In contrast, mutants F320M, S321M, L322M, V324M, and I330M produced only the ϳ50-kDa fragments (Fig. 7A, arrow c) that correspond to the shaded region in schematic diagram c (calculated peptide masses ϳ22.4 -23.3 kDa, with additional ϳ25 kDa of mass contributed from EL2 N-linked carbohydrate), with no production of fragments with masses of Ͻ8 kDa. Note that full-length DAT and the larger peptide fragments run with anomalously low electrophoretic mobility on these high percent gels and that more accurate mass estimates of the ϳ50-kDa CNBr fragment were obtained using lower concentration gels (63).
To verify that the 50-kDa fragments in these mutants originated from the indicated region, we performed deglycosylation analysis of selected mutants with neuraminidase and PNGase F, which cleave terminal sialic acids and N-linked carbohydrates from canonical N-glycosylation sites in EL2 (86,87). For fulllength WT, V324M, and I330M DATs (Fig. 7B, lanes 1, 5, and 9) treatment with neuraminidase plus PNGase F led to reductions in molecular mass due to deglycosylation (lanes 3, 7, and 11, and data not shown). As expected, deglycosylation did not alter the mass of the ϳ11-kDa WT CNBr fragment (Fig. 7B, lanes 2 and  4, arrow a), but it did reduce the masses of the ϳ50-kDa fragments from V324M and I330M DATs (lanes 6 and 10; arrow c) by ϳ25 kDa (lanes 8 and 12, arrow d). These results are consistent with the presence of EL2 in the ϳ50-kDa peptide fragments and further validate the indicated origins of the photolabeled fragments in the schematic diagrams.
The cleavage patterns shown in Fig. 7, A and B, indicate that ligand adduction occurs C-terminal to V318M and C319M and N-terminal to F320M, S321M, L322M, V324M, and I330M, and because CNBr proteolyzes peptide bonds on the C-terminal side of Mets, they demonstrate that the adduction of [ 125 I]RTI 82 occurs at Phe-320. These results also rule out a significant level of [ 125 I]RTI 82 adduction to other residues within this stretch of TM6, including Phe-326, an interaction that was suggested in a large portion of the decoys from RL docking to the outward-facing rDAT model.
Although CNBr proteolysis of [ 125 I]RTI 82-labeled F320M, S321M, L322M, V324M, and I330M did not produce the ϳ6-kDa fragments seen in V318M and C319M hDATs, some lightly labeled fragments of ϳ8 -10 kDa were seen in some digests. These fragments likely originate from adduction of [ 125 I]RTI 82 to a different region of the DAT primary sequence, as we have obtained preliminary evidence from antibody-based mapping that a small fraction of [ 125 I]RTI 82 adduction occurs C-terminal to TM6. 5 Multisite incorporation of DAT photoaffinity labels has been previously demonstrated (88) and likely occurs due to the proximity of the TM domains in the protein core in conjunction with small fluctuations in AIP moiety orientation during the photoactivation process. However, the significantly lower labeling intensity of this secondary site relative to that occurring in TM6 indicates that Phe-320 is the predominant site of adduction.

Analysis of RTI 82 Protection of S2 Residues from Inactivation by Methanethiosulfonate (MTS) Reagents-
The convergence of our computational docking and peptide mapping results strongly indicated that binding of the RTI 82 tropane pharmacophore occurs within the S1-binding site and positions the AIP group for adduction to Phe-319/320. However, Phe-319 is present at the interface between S1 and S2 (Fig. 8A), and thus RTI 82 tropane positioning in S2 could potentially also result in cross-linking of this site. To assess this possibility, we analyzed several residues present in S1 (yellow sticks), S2 (magenta sticks), or intermediate positions (green sticks) of the DAT per-5 R. A. Vaughan, unpublished data.  4 and 6, arrow b) that correspond to the shaded region in schematic diagram b. CNBr treatment of the remaining constructs produced fragments of ϳ50 kDa (lanes 8, 10, 12, 14, and 16, arrow c) that correspond to the shaded region in schematic diagram c but no fragments of Ͻ8 kDa. Filled circles in schematic diagrams represent Mets present in the constructs and the star represents the site of ligand adduction at Phe-320. B, equal amounts of radioactivity were subjected to the indicated combinations of neuraminidase plus PNGase F treatment (N/P) and CNBr digestion. Arrow a, CNBr fragment obtained from untreated and deglycosylated WT DAT; arrow c, CNBr fragments obtained from V324M and I330M DATs prior to deglycosylation; arrow d, CNBr fragments obtained from V324M and I330M DAT after deglycosylation. OCTOBER 24, 2014 • VOLUME 289 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29721 meation pathway (Fig. 8A) for cocaine-and RTI 82-induced protection from SCAM reagents. All mutants were made in an MTSET-insensitive rDAT E2C (C90A and C305A) background (89).

Tropane-binding Site on DAT
For transport protection experiments, we generated Cys mutations of residues Trp-84, Ile-159, Asp-475, and Ala-479, which are part of S2 or line the S1-S2 transition region (Fig. 8A, magenta and green sticks). All mutants were active, possessing DA transport activities ranging from 15 to 78% of the E2C DAT level. As expected, MTSET treatment of E2C DAT did not reduce uptake, consistent with the remaining Cys residues in the protein being inaccessible to the reagent. For protection FIGURE 8. SCAM protection analysis of S1 and S2 binding pockets. A, representation of Cys-engineered mutants in the S1 (yellow sticks) and S2 (magenta sticks) binding pockets as well as residues that reside in the interface between S1 and S2 (green sticks). The RosettaLigand pose for RTI 82 is shown (black sticks). B, HeLa cells transiently expressing the indicated DAT mutants were preincubated with vehicle or 5 M (Ϫ)-cocaine for 10 min followed by addition of 5 mM MTSET for 10 min. Cells were washed and incubated for 30 min at 37°C to release bound cocaine and analyzed for recovered activity by [ 3 H]DA transport assays. The samples labeled Control and Cocaine (white and black bars, respectively) were assayed following an initial wash and served as a control to verify cocaine inhibition of uptake. Results are normalized to percent activity of control, and the values represent the mean Ϯ S.E. from three independent experiments. MTS-treated samples with or without cocaine pretreatment were analyzed using two-way ANOVA with a post hoc Bonferroni test. ***, p Ͻ 0.001; n.s., not significant. C, immunoblots of the surface pool of DAT Cys mutants in the DAT E2C background purified with MTSEA-biotin in the absence (Ϫ) or presence of cocaine (ϩ, 100 M; ϩϩ, 1 mM (top panel)) or RTI 82 (ϩ, 10 M; ϩϩ, 50 M) (bottom panel). D, quantification of DAT bands in C and total DAT expression (data not shown) were determined using ImageJ (National Institutes of Health). Surface samples were normalized to total DAT and the surface cocaine-(black bars) or RTI 82 (red bars)-treated samples were expressed as percent of the respective untreated samples (white bars). The data represent three or more independent experiments. Paired t test analysis was used to determine significant differences between RTI 82-treated and -untreated samples. *, p Ͻ 0.05; **, p Ͻ 0.01. analysis, cells were co-incubated with cocaine prior to addition of MTSET. All DAT forms preincubated with cocaine showed recovery of transport activity after washing (wash control), demonstrating the reversibility of cocaine binding. MTSET treatment of W84C, I159C, D475C, and A479C DATs resulted in a 60 -90% loss in transport activity (Fig. 8B), indicating full or partial accessibility of these residues to modification. Co-incubation of the cells with (Ϫ)-cocaine during MTSET treatment did not protect the mutants from inactivation, indicating a lack of cocaine pharmacophore binding in S2. In addition, the sensitivity of I159C DAT to MTSET was enhanced compared with the non-cocaine-treated condition, suggesting that binding induced a conformational change that makes the I159C side chain more accessible to the reagent. We were unable, however, to assess RTI 82 protection of these residues by this method as RTI 82-pretreated cells recovered only a small amount of transport activity, even with extensive washing. This lower level of RTI 82 reversibility may be due to a slower release caused by hydrophobicity of the AIP or to other structural differences from (Ϫ)-cocaine related to its higher affinity. Furthermore, the impact of Cys substitution at S1 residues prevented accessibility testing by this method.
For these reasons, we assessed the ability of RTI 82 and cocaine to protect S1 and S2 residues by Cys-directed biotinylation. For these analyses, we generated additional S1 and S2 Cys mutations at Asp-79, Ala-81, Asn-82, Arg-85, Val-152, Phe-319, Ser-421, and Ile-483. All mutants were expressed as full-length proteins and were active for [ 3 H]CFT binding. MTSEA-biotin protection assays were done using 10 M RTI-82 or 100 M cocaine, except for D79C and S421C DATs, which exhibited a marked loss in potency to inhibit binding and required higher inhibitor concentrations (50 M RTI-82 or 1 mM cocaine) for protection analyses.
Surface rDAT was readily labeled with MTSEA-biotin, and this labeling was unaffected by addition of RTI 82 or cocaine (Fig. 8C, Control). Biotinylation of rDAT E2C was undetectable (Fig. 8C, Control), confirming its use as a suitable background for analysis of inserted Cys residues. Recovery of biotinylated rDAT surface protein from the S1 mutants D79C and S421C was significantly reduced in the presence of either RTI-82 or cocaine, indicating the protection of these residues from the MTS reagent (Fig. 8, C and D). V152C and N82C showed different patterns of reactivity, with V152C reactivity increased by cocaine but reduced by RTI-82, and N82C reactivity being increased by both cocaine and RTI 82 (Fig. 8, C and D). Val-152 is critical for high affinity antagonist binding and is thought to form important hydrophobic interactions with both substrates and inhibitors (15,37,38), and thus the slight differences in cocaine and RTI 82 structure apparently induce different alterations of the Cys side chain that results in the differential accessibility. Asn-82 is homologous to Asn-101 in human SERT, which is a structurally dynamic residue important in coupling to Na ϩ ions at the Na1 site (24,25), with our data supporting an increased accessibility of this site upon cocaine or RTI 82 binding. In contrast, neither RTI 82 nor cocaine altered the MTS accessibility of S2 residues W84C, R85C, I159C, and D475C (Fig. 8, C and D). Together, these findings strongly indicate that RTI-82 and cocaine bind to DAT in the S1 but not the S2 site.
Residues Phe-319, Ile-483, and Ala-479 lie at the interface between S1 and S2 (Fig. 8A, green sticks). Co-incubation with cocaine protected all of these sites from MTS modification, whereas RTI 82 protected only A479C (Fig. 8, C and D). This outcome is not unexpected given that the AIP group of RTI 82 is oriented toward the outer vestibule and may prevent complete transition to the occluded structure, leaving the residues accessible to modification. Cocaine lacks the AIP group and may thus induce fuller transition to the occluded structure that provides more complete protection of these residues, which could account in part for the differential effects of RTI 82 and cocaine on increased or decreased MTS accessibility in S1 residues N82C and V152C. In addition, we found that cocaine protected A479C from MTS attack in the MTSEA-biotin pulldown study (Fig. 8C) but not in the washout studies (Fig. 8B). This discrepancy may be due to bulk size difference between MTSEA-biotin and MTSET. These results suggest that the MTS accessibility of the intermediate residues may provide a readout for the S1 site on the transporter indicating whether a bound antagonist induces an occluded or more open conformation.

DISCUSSION
The kinetic interactions of DAT with cocaine and its analogs are complex, resulting in high and low affinity binding states (90,91) and competitive and noncompetitive uptake inhibition (92,93). We currently lack a complete mechanistic understanding of these properties, as some mutagenesis and comparative modeling studies support the interaction of cocaine at S1, where it could suppress transport by competing with substrate for key interactions (37,54), although other studies support binding near S2, where it could allosterically stabilize an inactive state of the transporter (94 -97). It has been proposed that these binding modes are not mutually exclusive and that cocaine may initially bind near the S2 site before transitioning to S1 following conformational changes (94,95). The current collection of LeuT and dDAT crystal structures supports multiple binding sites for substrates and inhibitors (15-19, 40, 42, 56, 60, 97, 98) and both allosteric and competitive inhibition mechanisms (6, 12, 17-20, 38, 41-43, 55, 60, 97, 99 -101). To date, however, no high resolution crystal structures of cocainebound SLC6 transporters have been obtained (6,12,102), and the exact cocaine binding site(s) on DAT and their relationships to the various kinetic states remain unresolved.
As a positive function approach to these issues, our groups have developed irreversible binding tropane and benztropine analogs as probes for physical identification of ligand interaction sites (63,80,103). Here, we provide the first identification of a specific photoaffinity analog contact point on DAT, and we used this information in conjunction with modeling and SCAM analysis to directly demonstrate binding of the tropane pharmacophore in the S1 substrate site. [ 125 I]RTI 82 labeling of DAT occurs with appropriate pharmacology (63), and because our photolabeling analyses utilized 5 nM ligand, the [ 125 I]RTI 82 adduction site identified in this study represents the high affinity, pharmacologically relevant antagonist-bound state of the transporter. It is likely that this site closely correlates with the (Ϫ)-cocaine-binding site, as the compounds share the N-methyl tropane pharmacophore that drives high affinity cocaine recognition (104) and differ only in the substitution of the 3␤-benzoyl ester with a phenyl chloride and addition of the 2␤-ethyl aryliodoazido moiety.
Several other lines of pharmacological and structural evidence support the validity of our findings and extend our understanding of the binding pocket. First, our top scoring models predict salt-bridge interaction between Asp-79 and the tropane nitrogen of RTI 82, consistent with the demonstrated requirement of this interaction in cocaine and CFT binding (31,37,49). During MD simulations of rDAT-RTI 82 complexes, we observed a slight downward translation of RTI 82 deeper into the S1 pocket, but importantly, the ligand maintained interaction with Asp-79, corroborating its importance in RTI 82 binding. This is likely to represent an important molecular motif in all NSS proteins, as interaction of N-based antagonists with the TM1 Asp residue has also been demonstrated in the dDAT and the SERT/LeuT hybrid transporter co-crystallized with bound SSRI and TCA inhibitors (40,56). A second important interaction was identified between the phenyl chloride of RTI 82 and Asn-157, which parallels the interaction of this residue with the phenyl fluoride of CFT (37). It is unclear whether Asn-157 would interact with cocaine, which possesses a nonhalogenated benzoyl ester at the 3␤ position and suggests this interaction as likely to contribute to the higher binding affinity of CFT and RTI 82 (104). In addition, residues homologous to Val-152 and Ser-421, which we found as contact points for RTI 82 binding, have been implicated in high affinity antagonist binding in SERT (38,55). Finally, in both RL and IFD, the RTI 82 poses we identified are virtually superimposable with respect to tropane pharmacophore orientation in the S1 pocket and extension of the arylazido arm out of the pocket toward the external vestibule. These findings are consistent with QSAR studies demonstrating that the tropane pharmacophore drives the major component of cocaine analog binding, and it provides a structural rationale for findings that bulky substituents can be accommodated at the 2␤ position of the tropane ring without significant impact on affinity (104).
The adduction site of RTI 82, Phe-319/320, functions as a gating residue on the outer margin of the S1 binding pocket and performs a crucial role in the transition between the "outward" and outward-occluded states by aromatic interaction with Tyr-156 (40). We obtained reasonable docked structures in both the open-to-out and outward-occluded models that identified Phe-319 as the RTI 82 adduction site; thus, it is possible that cocaine-like molecules suppress transport by inhibiting the transition from the outward state to the outward-occluded form or by stabilizing the outward-occluded state and preventing progression to the "inward-facing" structure. Here, our findings from RL docking could indicate that the outward-occluded structure better represents the native binding mode of RTI 82. However, other DAT inhibitors may stabilize distinct transporter conformations that could potentially confer different functional and behavioral outcomes (e.g. benztropines). Computational analysis by Beuming et al. (37) predicted Phe-320 interaction with the tropane ring of CFT, and the homologous residue in the LeuT/SERT hybrid (Phe-253) has been shown to participate in binding of SSRIs and TCAs (56). Our findings, however, did not indicate interaction of the RTI 82 tropane with Phe-319. This is likely due to the AIP moiety restricting reorientation of this residue and preventing its movement toward the S1 pocket and closer to the tropane pharmacophore (40). It is possible that by preventing closure of the aromatic lid, extensions to this arm of RTI 82 could stabilize outward-facing DAT conformations, which might result in different functional outcomes than the inhibitor-stabilized occluded form.
The direct identification of photoaffinity ligand adduction sites confers major advantages for computational analyses of ligand poses by dramatically reducing the conformational space that must be sampled during docking analysis and by providing experimentally determined ligand-protein contact points that can be used as absolute requirements by which recovered binding poses can be filtered. This significantly enhances the confidence with which poses can be interpreted and further analyzed by computational and mutational strategies to clarify overall transport and transport inhibition mechanisms. In addition, these procedures can be performed in functional mammalian transporters in which the WT form is directly compared with transport-and binding-competent mutant forms, which strongly increases the likelihood that physiologically relevant structures are being assessed. These multiple strengths support the utilization of this approach in further predictive docking analyses of these and other categories of DAT ligands. This is important because to date no SLC6 transporters have been cocrystallized with cocaine-like compounds or atypical DAT inhibitors and because the uncertain structural and functional relationships between mammalian transporters and the mutation-stabilized dDAT and LeuT/SERT hybrid homologs may obscure significant differences in the transport and inhibitor binding properties of the proteins (40,56).
The tremendous sociological and economical impacts of cocaine addiction make basic understanding of the mechanisms of cocaine binding and transport inhibition an important effort, especially with the promise afforded by the identification of the benztropine class of DAT antagonists, which bind to DAT and block DA uptake but do not produce the cocaine-like behavioral profiles and reduce cocaine self-administration in animal models (105,106). It has been suggested that the benztropines may bind distinct conformations of DAT and/or possess a slow rate of occupancy following administration, which may modulate the psychotropic effects of increased synaptic DA (11,37,105,106). Recently, modafinil and its R-enantiomer (armodafinil) have also been described as having distinctive interactions at DAT that might contribute to their nonaddictive and therapeutic profiles (96,(107)(108)(109). Thus, understanding cocaine binding in relation to compounds like these atypical DAT inhibitors could provide critical insights for developing medication strategies toward treating cocaine addiction.