Unbiased Simulations Reveal the Inward-Facing Conformation of the Human Serotonin Transporter and Na+ Ion Release

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 µs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na+-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport

Ligand Binding in the Extracellular Vestibule of the Neurotransmitter Transporter Homologue LeuT

The human monoamine transporters (MATs) facilitate the reuptake of monoamine neurotransmitters from the synaptic cleft. MATs are linked to a number of neurological diseases and are the targets of both therapeutic and illicit drugs. Until recently, no high-resolution structures of the human MATs existed, and therefore, studies of this transporter family have relied on investigations of the homologues bacterial transporters such as the leucine transporter LeuT, which has been crystallized in several conformational states. A two-substrate transport mechanism has been suggested for this transporter family, which entails that high-affinity binding of a second substrate in an extracellular site is necessary for the substrate in the central binding site to be transported. Compelling evidence for this mechanism has been presented, however, a number of equally compelling accounts suggest that the transporters function through a mechanism involving only a single substrate and a single high-affinity site. To shed light on this apparent contradiction, we have performed extensive molecular dynamics simulations of LeuT in the outward-occluded conformation with either one or two substrates bound to the transporter. We have also calculated the substrate binding affinity in each of the two proposed binding sites through rigorous free energy simulations. Results show that substrate binding is unstable in the extracellular vestibule and the substrate binding affinity within the suggested extracellular site is very low (0.2 and 3.3 M for the two dominant binding modes) compared to the central substrate binding site (14 nM). This suggests that for LeuT in the outward-occluded conformation only a single high-affinity substrate binding site exists

Solvent exposure of the aromatic lid in hSERT with noribogaine A), serotonin B) and cocaine C).

<p><b>A</b>)–<b>C</b>). Plots of the SASA of the side chains of the aromatic lid, Phe335 and Tyr176, as it evolves during the six trajectories for each ligand.</p

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

<div><p>The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from <i>Aquifex Aoelicus</i>. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.</p></div

Binding of serotonin to the extracellular binding pocket of hSERT.

<p>A representative pose of serotonin binding in the extracellular binding site as obtained from induced fit docking. Serotonin is shown in purple spheres, and noribogaine (in the central binding pocket) is shown in orange spheres. TM1 (red), TM3 (blue), TM6 (green) and TM8 (yellow) are shown in cartoon. The amino acid residues in the extracellular gate are shown in orange sticks. TM2, TM4, TM5, TM7 and TM9 are shown as beige cylinders.</p

Binding of Noribogaine, serotonin and cocaine to the central binding pocket of hSERT.

<p>The transmembrane helices that constitute the central binding site, TM1 (red), TM3 (blue), TM6 (green) and TM8 (yellow) are shown in cartoon and the side chains of central amino acid residues belonging to these helices are shown in grey sticks. Residues 171 to 174 in TM3 have been omitted for clarity. The ions are displayed as transparent spheres, the sodium ions in cyan and the chloride ion in yellow. <b>A</b>) The selected binding mode of noribogaine within the primary binding site in hSERT. The ligand is shown in orange sticks. <b>B</b>) Biochemically validated binding mode of serotonin (purple) in hSERT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063635#pone.0063635-Celik1" target="_blank">[12]</a>. <b>C</b>) Binding mode of cocaine (cyan) observed within the primary binding site of hSERT similar to the binding mode of cocaine in hDAT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063635#pone.0063635-Beuming2" target="_blank">[13]</a>.</p

Solvent accessibility of the intracellular pathway residues in hSERT.

<p><b>A</b>)–<b>C</b>)<b>.</b> Plots of the total SASA of the intracellular pathway residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063635#pone.0063635-Forrest1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063635#pone.0063635-Jacobs1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063635#pone.0063635-Zhang1" target="_blank">[70]</a> from the noribogaine, serotonin and cocaine systems, respectively.</p

Correlation between SASA of the intracellular pathway and the intracellular distance between the scaffold and bundle.

<p>The intracellular scaffold-bundle distance is plotted as a function of the calculated SASA of the intracellular pathway for <b>A</b>) noribogaine, <b>B</b>) serotonin and <b>C</b>) cocaine respectively. In all plots the LeuT crystal structures have been included as reference points; PDB code 2A65 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063635#pone.0063635-Yamashita1" target="_blank">[5]</a> in grey, PDB code 3TT1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063635#pone.0063635-Krishnamurthy1" target="_blank">[25]</a> as pale red, and PDB code 3TT3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063635#pone.0063635-Krishnamurthy1" target="_blank">[25]</a> in purple.</p

Ligand dependent bundle-scaffold interactions.

<p><b>A</b>) Noribogaine accommodates interactions between TM1 of the bundle (Asp98 (TM1) and Tyr 95 (TM1)) and the scaffold either through Ser438 (TM8) or Ala169 (TM3). <b>B</b>) Serotonin maintains stable interactions between the scaffold (Ser438 (TM8)) and both TM1 and TM6 (Asp98 (TM1), Tyr95 (TM1) and Phe335 (TM6)) in the bundle. <b>C</b>) Cocaine only interacts with TM1 (Asp98) and TM6 (Phe335) of the bundle.</p