Y95 and E444 interaction required for high-affinity S-citalopram binding in the human serotonin transporter

Abstract

The human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT) is responsible for the reuptake of 5-HT following synaptic release, as well as for import of the biogenic amine into several non-5-HT synthesizing cells including platelets. The antidepressant citalopram blocks SERT and thereby inhibits the transport of 5-HT. To identify key residues establishing high-affinity citalopram binding, we have built a comparative model of hSERT and Drosophila melanogaster SERT (dSERT) based on the Aquifex aeolicus leucine transporter (LeuT(Aa)) crystal structure. In this study, citalopram has been docked into the homology model of hSERT and dSERT using RosettaLigand. Our models reproduce the differential binding affinities for the R- and S-isomers of citalopram in hSERT and the impact of several hSERT mutants. Species-selective binding affinities for hSERT and dSERT also can be reproduced. Interestingly, the model predicts a hydrogen bond between E444 in transmembrane domain 8 (TM8) and Y95 in TM1 that places Y95 in a downward position, thereby removing Y95 from a direct interaction with S-citalopram. Mutation of E444D results in a 10-fold reduced binding affinity for S-citalopram, supporting the hypothesis that Y95 and E444 form a stabilizing interaction in the S-citalopram/hSERT complex.

Keywords: S-citalopram; computational docking; hSERT; homology model; ligand.

Figure 1

(A) S-Citalopram functional groups involved in binding are labeled a−f. (B) Contacts within the S-citalopram/hSERT high-affinity binding mode are displayed as a heatmap. Given is the fraction of models that display a contact within the largest cluster of models. Residues known to affect S-citalopram binding are shown within the black box.

Figure 2

S- and R-citalopram in complex with mutants of hSERT. The extracellular side of the protein is shown on the top of all images, whereas the cytoplasm is at the bottom of all images. The S-citalopram/hSERT complex is shown on top, and the R-citalopram/hSERT complex is shown below. Experimental binding affinities, K_i, and computationally predicted binding energies are given below each image. (A and E) WT hSERT in complex with S- and R-citalopram. Experimentally verified residues involved in binding are shown as sticks and highlighted in red and labeled. (B and F) S- and R-citalopram putative wild type binding mode docked into the I172M mutant of hSERT (green). The original wild type binding mode is displayed in yellow and blue (S-citalopram and R-citalopram, respectively). The mutation I172M is shown in green and stick format with the experimentally verified residues shown in cartoon and highlighted with red. (C and G) S- and R-citalopram docked into the Y95F mutant. The putative wild type binding is colored cyan with the mutant Y95F colored in cyan and shown as a stick. (D and H) S- and R-citalopram docked into S438T. The putative wild type binding is colored in orange with the mutant S438T shown in sticks and colored orange.

Figure 3

(A) Binding mode of the S-citalopram/hSERT complex with the depiction of the Y95 (gray) in a downward position. Mutation of E444D (not shown) results in Y95 populated in two positions, a downward position (gray) and an upward position (pink). (B) Mutation of E444D results in a 10-fold loss of potency for S-citalopram suggesting that the Y95 switches between two different conformations: upward (pink) and downward conformations (gray). The wild type is shown in by black squares and a line, and E444D is shown in circles and spheres.

Figure 4

Putative binding mode of the S-citalopram/dSERT complex. S-Citalopram is shown in white. Residues that contribute to lowering the energy of the complex are highlighted in red. Substitution of A169 to D164 dSERT results in a higher solvation score for the complex. Additionally, M167 dSERT results in an increase in the VDW potential.