Substrate binding and translocation of the serotonin transporter studied by docking and molecular dynamics simulations

Abstract

The serotonin (5-HT) transporter (SERT) plays an important role in the termination of 5-HT-mediated neurotransmission by transporting 5-HT away from the synaptic cleft and into the presynaptic neuron. In addition, SERT is the main target for antidepressant drugs, including the selective serotonin reuptake inhibitors (SSRIs). The three-dimensional (3D) structure of SERT has not yet been determined, and little is known about the molecular mechanisms of substrate binding and transport, though such information is very important for the development of new antidepressant drugs. In this study, a homology model of SERT was constructed based on the 3D structure of a prokaryotic homologous leucine transporter (LeuT) (PDB id: 2A65). Eleven tryptamine derivates (including 5-HT) and the SSRI (S)-citalopram were docked into the putative substrate binding site, and two possible binding modes of the ligands were found. To study the conformational effect that ligand binding may have on SERT, two SERT-5-HT and two SERT-(S)-citalopram complexes, as well as the SERT apo structure, were embedded in POPC lipid bilayers and comparative molecular dynamics (MD) simulations were performed. Our results show that 5-HT in the SERT-5-HT(B) complex induced larger conformational changes in the cytoplasmic parts of the transmembrane helices of SERT than any of the other ligands. Based on these results, we suggest that the formation and breakage of ionic interactions with amino acids in transmembrane helices 6 and 8 and intracellular loop 1 may be of importance for substrate translocation.

Graphical abstract

SERT–5-HT^B binding mode

Fig. 1

Ligand binding modes detected through docking. a SERT–5-HT^A binding mode, b SERT–5-HT^B binding mode, c SERT–(S)-citalopram^A binding mode, and d SERT–(S)-citalopram^B binding mode. The side chains of amino acids Y95, D98 and I172 and the binding pocket detected by ICM PocketFinder (red wire representation) are shown. Color coding of atoms in amino acids: red oxygen, blue nitrogen, gray carbon and hydrogen. Color coding of ligands: red oxygen, blue nitrogen, yellow carbon, gray hydrogen

Fig. 2

SERT structures. a Initial SERT structure and b the average SERT–5-HT^B structure generated based on the last 10 ns of the MD simulation. “Intra-structural” pockets detected by ICM PocketFinder are shown. The putative substrate binding pocket is represented as red wire

Fig. 3

a Intracellular view of the average SERT–5-HT^B structure. SERT Cα carbon atoms are shown in gray cylindrical representation. For clarity, amino acids 148–160, 338–350 and 444–453 are shown in blue. The putative substrate binding site is displayed as red wire. Amino acids that are proposed to play a role in the opening of a vestibule extending from the putative substrate binding site (red wire representation) to the cytoplasm are shown as xstick. b Close-up of a with residues in xstick. Green lines show interactions formed during the simulation; red line shows an interaction broken during simulation

Fig. 4

Comparison of the 5-HT binding mode in the initial SERT–5-HT^B complex (gray) and that in the average SERT–5-HT^B structure generated based on the last 10 ns of MD (orange). Atomic distances (Å) are shown as dotted lines. For clarity, selected hydroxyl oxygen atoms on 5-HT, Y95 and G338 are colored red