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

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

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

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

Movement of the bundle with respect to the scaffold.

<p><b>A</b>) The scaffold together with TM11 and TM12 are shown in grey. TM10 and TM11 are transparent for clarity. TM1 (dark red), TM2 (pink), TM6 (green) and TM7 (green) correspond to the bundle in hSERT. The extracellular part of the scaffold was defined as the center of mass of the C_α atoms of the third and fourth extracellular residues of TM3, TM4, TM5, TM8, TM9, and TM10 at the extracellular side, with the bundle part defined similarly as the center of mass of the C_α atoms in the fourth and third extracellular residues of TM1, TM2, TM6, and TM7. These two centers of mass for the bundle and scaffold are shown as yellow balls connected by a line. The intracellular scaffold-bundle distance is similarly defined from the corresponding intracellular residues and the intracellular scaffold-bundle distance is illustrated by cyan spheres connected by a line. <b>B</b>) –<b>D</b>) Boxplots of the extracellular scaffold-bundle distance. The region corresponding to 50% of the data points is shown as a box and the minimum and maximum values are represented by whiskers. <b>E</b>) –<b>G</b>) Boxplots of the intracellular scaffold-bundle distance.</p

Measurements of the intracellular gating network in hSERT with noribogaine (A), serotonin (B) and cocaine (C).

<p><b>A</b>)–<b>C</b>)<b>.</b> Plot of the shortest distance between the oxygen atom of the hydroxyl of Tyr350 and the carboxylate oxygen atoms in Glu444 as it evolves during the simulations. The grey box highlights the area from 2.5–4 Å.</p

Regulation of the Ca²⁺-ATPase by cholesterol: A specific or non-specific effect?

<p>Like other integral membrane proteins, the activity of the Sarco/Endoplasmic Reticulum Ca²⁺-ATPase (SERCA) is regulated by the membrane environment. Cholesterol is present in the endoplasmic reticulum membrane at low levels, and it has the potential to affect SERCA activity both through direct, specific interaction with the protein or through indirect interaction through changes of the overall membrane properties. There are experimental data arguing for both modes of action for a cholesterol-mediated regulation of SERCA. In the current study, coarse-grained molecular dynamics simulations are used to address how a mixed lipid-cholesterol membrane interacts with SERCA. Candidates for direct regulatory sites with specific cholesterol binding modes are extracted from the simulations. The binding pocket for thapsigargin, a nanomolar inhibitor of SERCA, has been suggested as a cholesterol binding site. However, the thapsigargin binding pocket displayed very little cholesterol occupation in the simulations. Neither did atomistic simulations of cholesterol in the thapsigargin binding pocket support any specific interaction. The current study points to a non-specific effect of cholesterol on SERCA activity, and offers an alternative interpretation of the experimental results used to argue for a specific effect.</p