Structural basis for molecular recognition at serotonin receptors

Abstract

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

Fig. 1

Overall architecture of the 5-HT_1B receptor bound to ERG and comparison of the ligand binding pocket shapes of the 5-HT_1B receptor and the 5-HT_2B receptor. (A) The 5-HT_1B receptor is shown as a light blue colored ribbon cartoon, with N terminus, ICL1 and ICL2 highlighted in yellow. Ligand ERG is colored magenta. The disulfide bond between C122 and C199 is shown as orange sticks. The Y40 and D352^7.36 side chains, which mediate the interaction between the N terminus and the ligand binding pocket, are shown as green sticks with a dashed line indicating the hydrogen bond interaction. L80^ICL1 and Y157^ICL2 interact with residues of the 7TM bundle stabilizing the local structures of ICL1 and ICL2. (B) Shown at the bottom is the superposition of the 5-HT_1B/ERG structure (light blue) and the 5-HT_2B/ERG structure (white). The ligands are colored magenta for the 5-HT_1B receptor and green for the 5-HT_2B receptor. The top panel shows an extracellular view of the ligand binding sites. The arrow indicates a 3.0 Å shift (distance measured between the α-carbons of T209^5.39 in the 5-HT_1B receptor and M218^5.39 in the 5-HT_2B receptor) at the extracellular end of helix V. (C) and (D) The surface representation of the ligand binding pockets of the 5-HT_1B receptor and the 5-HT_2B receptor are shown in transparent pink and transparent green, respectively.

Fig. 2

Comparison of ligand-receptor interactions in 5-HT_1B/ERG and 5-HT_2B/ERG structures. (A), (B) and (C) Superposition of the ligand binding pockets of 5-HT_1B receptor (light blue) and 5-HT_2B receptor (white). The carbons of ligand ERG in the 5-HT_1B and the 5-HT_2B receptor structures are shown as magenta and green, respectively. (A) Residues forming the orthosteric binding sites are shown as sticks and labeled in blue for 5-HT_1B receptor and black for 5-HT_2B receptor. The salt bridge interactions between D^3.32 and ERG, as well as the hydrogen bond interactions between T^3.37 and ERG, Y^7.43 and D^3.32, are shown as red dashed lines for 5-HT_1B receptor and green dashed lines for 5-HT_2B receptor. (B) Substantial differences in the extended ligand binding pockets resulted in different conformations of the ligand ERG. (C) The overall binding pockets with the orthosteric and the extended ligand binding sites shaded red and blue, respectively. (D) and (E) Diagram representation of ligand interactions in the binding pockets of 5-HT_1B and 5-HT_2B receptors, respectively. Intramolecular hydrogen bonds within the cyclic tripeptide moiety are indicated as dashed lines. Residues in the orthosteric binding pockets are shown in red boxes, while extended binding pocket residues are shown in blue boxes. The hydrogen bond interaction between T^3.37 and ERG and the salt bridge interactions between D^3.32 and ERG are indicated by red dashed lines. In (A), (B) and (D), Y208^5.38 and P338^6.59 which do not interact with ERG are labeled in grey.

Fig. 3

Docking of the promiscuous (5-HT and LSD) and selective (sumatriptan and norfenfluoramine) ligands into the binding pockets of the 5-HT_1B and 5-HT_2B receptor structures. Docking of 5-HT (A), LSD (B), sumatriptan (C) and norfenfluramine (D) into the orthosteric binding pockets of 5-HT_1B (receptor colored light blue; ligands colored magenta) and 5-HT_2B (receptor colored white; ligands colored green) receptors. In (A) and (B), the polar interactions between Y^7.43 and D^3.32, and between D^3.32, T^3.37 with the ligands are shown as dashed lines. In (A), the non-polar interactions between the indole ring of 5-HT and residues at positions 5.42 and 5.46 are shown as dotted lines. In (C), steric hindrance from M218^5.39 forced a reorientation of the sulfonamide group and a shift of the indole core structure of sumatriptan when docked into 5-HT_2B receptor. In (D), in the 5-HT_2B receptor, F217^5.38 and M218^5.39 form closed contacts with the trifluoromethyl group of the ligand, which are absent in 5-HT_1B receptor.

Fig. 4

Structural basis for differences in the pharmacological properties between human and rodent 5-HT_1B receptors. The high affinity β-adrenergic antagonists propranolol (A) and cyanopindolol (B), both shown in yellow colored carbons, are docked in the model based on the human 5-HT_1B/ERG structure with T^7.39 mutated to N, as found in 5-HT_1B rat and mouse orthologs. The N^7.39 side chain (magenta carbons) remained flexible in the docking procedure. The hydrogen bond network involving N^7.39, D^3.32 and Y^7.43 and propanolamine moieties of the ligands is shown as orange dots. Carazolol (green carbons) from the superimposed β₂-adrenergic receptor structure (PDB ID: 2RH1) is shown for comparison.