A Gating Mechanism of the Serotonin 5-HT3 Receptor Shuguang Yuan, Slawomir Filipek, Horst Vogel  Structure  Volume 24, Issue 5, Pages 816-825 (May 2016) DOI: 10.1016/j.str.2016.03.019 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 W156 Conformational Switches and Interaction Network between Residues in the 5-HT3R (A) Serotonin in the “aromatic cage” (dotted circle) at the interface between two neighboring extracellular domains. (B) Configuration of serotonin in the aromatic cage of the 5-HT3R after 30 ns of MD equilibration. (C) Configuration of serotonin in the aromatic cage of the 5-HT3R after 700 ns of MD simulation. Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 The Serotonin Binding Pocket of the 5-HT3 Receptor Residues lining the serotonin-binding pocket of the 5-HT3R are depicted, together with a conserved residues logo based on multiple sequence alignment on all cationic Cys-loop receptors, including four 5-HT3 serotonin receptors, 17 nicotinic acetylcholine receptors, and one zinc-activated ion channel. The presence of conserved aromatic residues including W63, Y126, W156, and Y207 imply that π-π interactions play a central role for binding of the neurotransmitter. Furthermore, the conserved negatively charged residues at position 209 indicate the importance of electrostatic interactions for the binding of serotonin in the 5-HT3R and beyond for ligand binding in the case of the other anionic Cys-loop receptors. The size of the single letters in the depicted sequence segments scales with its conservation at this position. Conserved residues participating in binding serotonin are highlighted by dashed rectangles. Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Simultaneous View of Community Residue Interaction Network and 3D Structure of the Neurotransmitter Binding Pocket of the 5-HT3R (A) Apo form without serotonin. (B) With bound serotonin. Colors of the dots in the community analysis correspond to colors in the indicated regions of the 3D protein structure. Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Domain Movements and Hydrophobic Pore of 5-HT3R (A) The normal modes of collective motions were calculated by principal component analysis. The sizes of the red arrows scale with the amplitudes of the particular motions. (B) Global twisting of the receptor's extracellular domain by Δφ = 5°–7°, indicated by overlaying the structure of the 5-HT3R before (bold) and after (faint) binding of serotonin. (C) Tilting of the β sandwich by Δθ = 5°–7°. Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Flexibility Pattern of a 5-HT3R Subunit and Cross-Sectional View of the 5-HT3R Channel before and after Activation by Serotonin (A) Flexibility of particular regions in a single submit are indicated by wider radius of tube and a red color. The most flexible regions are colored red, medium flexible regions white, and least flexible regions green. (B) Cross section of 5-HT3R in the non-activated state before binding of serotonin (left panel) and after binding of serotonin reaching an open, water-filled channel state (right panel). Hydrophobic regions are colored in orange and hydrophilic regions in cyan. (C) Pore diameter along z axis of the 5-HT3R for the non-activated receptor state (two independent MD simulations, black and cyan) and for the open, water-filled channel state (two independent MD simulations, red and blue). Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 The Hydrophobic Channel Gate (A) Comparison of dimensions of the hydrophobic constriction sites (channel gates) of the 5-HT3R in the closed-channel state (PDB: 4PIR) with that of GLIC in the open-channel state (PDB: 3P50). Shown are cross sections of the M2 TM helix of 5-HT3R (green) and GLIC (gray). Five L260 and five V264 residues (green) in the 5-HT3R were superimposed with corresponding residues I233 and A237 (purple) in GLIC. (B) Left: free-energy changes in the conformational space of the dihedral angles χ1 of L260 and V264 show two distinct minima of the closed (I) and open (II) channel state in the 5-HT3R. Right: distinct side-chain conformations of V264 (top) and L260 (bottom) of the 5-HT3R in the closed-channel state before binding of serotonin (I, green) and in the activated channel state after binding of serotonin (II, cyan). Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 The hydrophobic Gate of the 5-HT3 Receptor (A) The cross section perpendicular to the membrane plane through the A and D subunits of the pentameric 5-HT3R (left panel) in the crystal structure in the absence of serotonin reveals a closed hydrophobic gate formed by the L260 and V264 residues (cross sections at L260 and V264 planes, right panel) preventing the entrance of water molecules. Amino acid residues of the M2 helices along the channel are indicated. (B) A continuous water channel was formed by opening the gate of 5-HT3R by movements of side chains of L260 and V264. Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 8 Gating Mechanism of 5-HT3R Side-view cross section through the 5-HT3R shows two protein subunits with two bound serotonin molecules near the W156 residues in the aromatic ligand-binding cages. The global quaternary motions (black arrows) induce (1) the entrance of water molecules (red) into the central transmembrane channel across the hydrophobic gate represented by V264 and L260, (2) a deeper penetration of sodium ions (blue), and (3) the entrance of chloride ions (green) into the intracellular vestibule via lateral portals. Structure 2016 24, 816-825DOI: (10.1016/j.str.2016.03.019) Copyright © 2016 Elsevier Ltd Terms and Conditions