Structural insights into the ligand binding and Gi coupling of serotonin receptor 5-HT5A

Abstract

5-hydroxytryptamine receptor 5A (5-HT_5A) belongs to the 5-HT receptor family and signals through the G_i/o protein. It is involved in nervous system regulation and an attractive target for the treatment of psychosis, depression, schizophrenia, and neuropathic pain. 5-HT_5A is the only G_i/o-coupled 5-HT receptor subtype lacking a high-resolution structure, which hampers the mechanistic understanding of ligand binding and G_i/o coupling for 5-HT_5A. Here we report a cryo-electron microscopy structure of the 5-HT_5A-G_i complex bound to 5-Carboxamidotryptamine (5-CT). Combined with functional analysis, this structure reveals the 5-CT recognition mechanism and identifies the receptor residue at 6.55 as a determinant of the 5-CT selectivity for G_i/o-coupled 5-HT receptors. In addition, 5-HT_5A shows an overall conserved G_i protein coupling mode compared with other G_i/o-coupled 5-HT receptors. These findings provide comprehensive insights into the ligand binding and G protein coupling of G_i/o-coupled 5-HT receptors and offer a template for the design of 5-HT_5A-selective drugs.

Fig. 1. Cryo-EM structure of the 5-CT–5-HT_5A–G_i–scFv16 complex.

a Schematic illustration of 5-HT receptors coupling to distinct G proteins upon stimulation by 5-HT and 5-CT. 5-HT₁ and 5-HT₅ belong to G_i/o-coupled 5-HT receptors. 5-HT₁ subfamily includes 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, and 5-HT_1F. Different from other 5-HT receptor subtypes, 5-HT₃ is an ion channel. b, c Orthogonal views of the density map (b) and model (c) for the 5-CT–5-HT_5A–G_i–scFv16 complex. The EM density of 5-CT is shown as cyan surface presentation. d The extracellular view of the 5-HT_5A. The complex is colored by subunits. Light pink, 5-HT_5A; cyan, 5-CT; blue, Gα_i; salmon, Gβ; light green, Gγ; light blue, scFv16.

Fig. 2. The 5-CT-binding pocket of 5-HT_5A.

a Detailed interactions of 5-CT with residues in the binding pocket of 5-HT_5A. b 2D schematic representation of interactions between 5-CT and residues in the ligand-binding pocket of 5-HT_5A, analyzed by LigPlot⁺ program. The polar interactions are indicated as black dashed lines. c Effects of alanine mutation of pocket residues of 5-HT_5A on 5-CT-induced G_i protein recruitment. Three independent NanoBiT assays in triplicates were performed. Each data point presents mean ± SEM from a representative experiment.

Fig. 3. Role of the residue at 6.55 in determining 5-CT selectivity of G_i/o-coupled 5-HT receptors.

a Cross-section of the orthosteric binding pocket of 5-HT_5A. The hydrophobic interaction between 5-CT and E305^6.55 is indicated as a black dashed line. b 5-CT activity (pEC₅₀ values) for wild-type (WT) and mutants of G_i/o-coupled 5-HT receptors. ^#5-CT activity (pEC₅₀ values) for WT receptors. U.D., undetectable. c Effects of mutations at 6.55 on 5-CT-induced G_i protein recruitment of G_i/o-coupled receptors. Three independent NanoBiT assays in triplicates were performed. Each data point presents mean ± SEM from a representative experiment.

Fig. 4. Structural features of the activation and G_i/o coupling of 5-HT_5A and other G_i/o-coupled 5-HT receptors.

a The activation of 5-HT_5A and other G_i/o-coupled 5-HT receptors. The outward movement of TM6 of agonist-bound G_i/o-coupled 5-HT receptors compared with methiothepin-bound 5-HT_1B in the inactive state is shown as a black arrow. The movements of the αN and α5 helix of Gα_i protein in the 5-HT_5A–G_i complex relative to that in other G_i/o-coupled 5-HT receptors are indicated as black arrows. The structures of the active 5-HT_5A (light pink), 5-HT_1A (cyan, PDB: 7E2Y), 5-HT_1B (purple, PDB: 6G79), 5-HT_1D (green, PDB: 7E32), 5-HT_1E (wheat, PDB: 7E33), 5-HT_1F (coral, PBD: 7EXD) and inactive-state 5-HT_1B (gray, PDB: 4IAQ) complexes were superimposed based on TM2, TM3 and TM4. b Structural comparison of ligands and the W^6.48 residues of active 5-HT receptors with that of inactive 5-HT_1B. Ligands directly contact W^6.48. Compared with methiothepin, the inverse agonist of 5-HT_1B, agonists trigger the rotameric switch of W^6.48, which leads to the outward movement of TM6. c The sequence alignment of receptor residues at receptor–G_i/o protein interfaces. The conserved residues are highlighted in solid green circles. d, e Detailed interactions between 5-HT_5A and the Gα_i subunit. Detailed Interactions between TM5, TM6, and the TM7–helix 8 junction of 5-HT_5A and the α5 helix of the Gα_i subunit (d). Detailed interactions between ICL2 of 5-HT_5A and the α5 and αN helices, β1 and β3 strands of the Gα_i subunit (e).