Antidepressant specificity of serotonin transporter suggested by three LeuT-SSRI structures

Abstract

Sertraline and fluoxetine are selective serotonin re-uptake inhibitors (SSRIs) that are widely prescribed to treat depression. They exert their effects by inhibiting the presynaptic plasma membrane serotonin transporter (SERT). All SSRIs possess halogen atoms at specific positions, which are key determinants for the drugs' specificity for SERT. For the SERT protein, however, the structural basis of its specificity for SSRIs is poorly understood. Here we report the crystal structures of LeuT, a bacterial SERT homolog, in complex with sertraline, R-fluoxetine or S-fluoxetine. The SSRI halogens all bind to exactly the same pocket within LeuT. Mutation at this halogen-binding pocket (HBP) in SERT markedly reduces the transporter's affinity for SSRIs but not for tricyclic antidepressants. Conversely, when the only nonconserved HBP residue in both norepinephrine and dopamine transporters is mutated into that found in SERT, their affinities for all the three SSRIs increase uniformly. Thus, the specificity of SERT for SSRIs is dependent largely on interaction of the drug halogens with the protein's HBP.

Figure 1. Interaction of three SSRIs with LeuT and the crystal structures of their complexes

a. Binding of SSRIs to LeuT in detergent solution was measured using a scintillation proximity assay. The IC₅₀ values for sertraline, R-fluoxetine and S-fluoxetine to inhibit [³H]leucine binding to LeuT were determined to be 19.7 ± 9.2 μM, 2.54 ± 0.41 mM and 355 ± 46 mM, respectively. Curves show relative [³H]leucine binding, normalized to the [³H]leucine binding in the absence of inhibitors. Each point represents the mean ± S.E. (N = 3). b. LeuT transport activity measured in the absence and presence of SSRIs was measured in reconstituted proteoliposomes. Leucine transport by LeuT was completely inhibited by sertraline, R-fluoxetine and S-fluoxetine at concentration of 0.5 mM, 5 mM and 0.5 mM, respectively (N = 3). c. F_o-F_c simulated annealing omit maps of the SSRI from the three LeuT-SSRI complex structures, LeuT-sertraline at 2.15 Å resolution, LeuT-R-fluoxetine at 2.35 Å, and LeuT-S-fluoxetine at 2.45 Å resolution. The maps are contoured at 3 σ. d. Structure of the drug-binding site in the LeuT-sertraline complex at 2.15 Å resolution, viewed from within the membrane plane. e. Structure of the drug-binding site in the LeuT-R-fluoxetine complex at 2.35 Å resolution. f. Structure of the drug-binding site in the LeuT-S-fluoxetine complex at 2.45 Å resolution. The sertraline molecule is colored yellow, R-fluoxetine orange, and S-fluoxetine green. In e – f. helix TM11 in LeuT is omitted from the figures for clarity.

Figure 2. Comparison of common features of SSRI binding to LeuT and key determinants for specificity for SSRIs

a and b. Superposition of the three LeuT-SSRI structures at the drug binding site. c. Surface presentation of the halogen-binding pocket in the LeuT-S-fluoxetine structure . d. Illustration showing halogen substitutions at one or both of the 3rd and 4th positions in the phenyl ring of aminotetraline with Cl- or CF₃- yield an SSRI. e. Illustration showing phenoxyphenylpropylamine-based antidepressants. Substitutions at the 4- substitution in the phenoxy ring with F-, Cl-, CF₃-, CH₃- or OCH₃- produce SSRIs, including R- and S-fluoxetines, whereas substitutions at the 2nd position with CH₃- or OCH₃- yield NRIs like tomoxetine and nisoxetine . In all the panels, the SSRIs are colored as in Fig.1.

Figure 3. Halogen-binding pocket in SERT and its critical importance in the protein's specificity for antidepressant

a. Sequence alignment at the HBP between NSS proteins. The only difference in primary sequence between SERT and NET or DAT is at Gly100 in SERT, which is an alanine in the latter two proteins. b. Docking of R-fluoxetine to a SERT homology model . Only the trifluoromethylphenoxy group of the drug is shown for clarity. c – e. Changes in affinity (IC₅₀) for antidepressants of human SERT, NET and DAT when a residue at their HBP is mutated, relative to the appropriate wild-type transporter, as measured in binding assays with HEK293 cells. c. Binding of [3H]citalopram to the SERT-Ile179Asp mutant in the presence of sertraline, chlomipramine and desipramine. d. Binding of [3H]CFT to the NET-Ala77Gly mutant and to the DAT-Ala81Gly mutant (e) both in the presence of sertraline, R-fluoxetine and S-fluoxetine. 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).