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

Structure and mechanism of Zn^(2+)- transporting P-type ATPases

Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis. In prokaryotes and photosynthetic eukaryotes, Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2·P_i) of ZntA from Shigella sonnei, determined at 3.2 Å and 2.7 Å resolution, respectively. The structures reveal a similar fold to Cu^+-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn^(2+) ions by the transporter. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including C392, C394 and D714. The pathway closes in the E2·P_i state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn^(2+) release as a built-in counter ion, as has been proposed for H^+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter transport. These findings suggest a mechanistic link between P_(IB)-type Zn^(2+)-ATPases and P_(III)-type H^+-ATPases and at the same time show structural features of the extracellular release pathway that resemble P_(II)-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca^(2+)-ATPase (SERCA) and Na^+, K^+-ATPase. These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine

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

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

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

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