Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor

Abstract

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT_2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.

Keywords: GPCR; LSD; psychedelic; sertotonin receptor; signal transduction; structural biology.

Figure 1.. The Overall Cryo-EM Structure of the HTR2A-Gαq Bound to 25CN-NBOH and Crystal Structures of the HTR2A Bound to LSD or Methiothepin

(A) Cartoon view of the cryo-EM structure of HTR2A (green color) with agonist, 25CN-NBOH (yellow stick and 2D chemical structure, left), mini-Gαq (olive color), Gβ (red color), and Gγ (yellow color) with scFv16 omitted. See Figures S1 and Table S1. (B) Cartoon view of the crystal structure of HTR2A (pink color) with the partial agonist LSD (light blue stick and 2D chemical structure, left). (C) Cartoon view of the crystal structure of HTR2A (dark blue color) with the antagonist methiothepin (orange stick and 2D chemical structure, left). See Figure S2 and Table S1. (D) Schematic diagram of the bioluminescence resonance energy transfer (BRET) assays, BRET 1 and BRET 2. (E) The comparison of mini-Gαq or Gαq wild-type interactions with HTR2A upon the agonist 25CN-NBOH stimulation; data represent mean ± SEM of three biological replicates. See Table S4 for fitted parameter values. (F) Ligand-mediated mini-Gαq recruitment to HTR2A. Several hallucinogen agonists were compared in BRET 1 assays. See Table S4 for fitted parameter values. See also Figure S4 and Table S2.

Figure 2.. Overall Conformational Differences in the Ligand-Binding Pockets of 25CN-NBOH and LSD-Bound HTR2A

(A) View of the HTR2A ligand-binding pocket from the extracellular side. Conformational changes in cylindric helix and loop positions due to receptor activation are highlighted by red arrows. Distances were measured between the Cα atoms of I210^4.60, L229^ECL2, V235^5.39, N343^6.55, and E355^7.38. (B and C) Electrostatic surface representation from the extracellular view of HTR2A/LSD and HTR2A/25CN-NBOH, respectively. Electrostatic potential surfaces were calculated using the APBS plugin in PyMOL (Baker et al., 2001). (D) Expansion of binding pocket of HTR2A bound to full agonist 25CN-NBOH compared to partial agonist LSD. See Table S3 for volume estimates.

Figure 3.. Ligand-Specific Interactions with HTR2A

(A–C) Specific residues in the binding pockets that interact with 25CN-NBOH (yellow) (A), LSD (light blue) (B), and methiothepin (brown) (C), respectively. Alternative 2D diagrams showing direct interactions with each ligand are also provided at the bottom of each panel. The salt bridge interaction, as well as hydrogen bond interactions, is shown as red dashed lines. (D) Mutagenesis studies showing the effects of orthosteric-site residues on ligand-binding affinity and functional activity. Heatmap of ΔpEC₅₀ (EC_50WT-EC_50mt) (by BRET 2, HTR2A/Gαq) and ΔpKi (Ki_wt-Ki_mt) (by binding assay, [³H]-LSD) shows differences between HTR2A wild-type and mutants. See Figure S6 and Tables S5 and S6 for fitted parameter values that represent mean ± SEM of n = 3 biological replicates. See also Figure S5.

Figure 4.. Differential 25-CN-NBOH and LSD Binding Modes

(A) The superimposed structures of HTR2A with 25CN-NBOH (yellow), LSD (light blue), methiothepin (orange), risperidone (pink, PDB: 6A93), and zotepine (green, PDB: 6A94). (B) S159, W336, and G369 form a binding pocket that is important for 25CN-NBOH’s agonist activity. (C) W336^6.48 acts as a pivot for the outward movement of TM6. (D) Conformational displacement of side-chain of W336^6.48, followed by F332^6.44 in the P-I-F motif. See Figure S4. (E)The sequence alignment of the serotonin receptor family with an HTR2A specific residue S242^5.46 is highlighted. (F)The S242A^5.46 mutation accelerates LSD dissociation from HTR2A; data represent mean ± SEM of n = 3 biological replicates. (G)The overall structural comparison of HTR2A/LSD (pink/magenta color) and HTR2B/LSD (olive/lime color) and inset shows side view of LSD (magenta)-bound HTR2A (pink) crystal structure overlaid with the LSD (lime)-bound HTR2B (olive) structure. Hydrogen-bond interactions are highlighted by red dash lines. See also Figure S5 and S7.

Figure 5.. The Interface between the HTR2A and Gαq Protein and Confirmation of Its Functional Relevance

(A) The close up view of interaction of HTR2A (green color) and H5 helix (olive color) of Gαq protein. All residues involved in interaction show stick model. Hydrogen bond interactions are highlighted by the red dashed line. (B) The detailed view of interaction of ICL2 of HTR2A and Gαq and showing I181^ICL2 of HTR2A and its surrounded hydrophobic residues of Gαq. (C) BRET validation of residues in the HTR2A interface. See Table S7 for fitted parameter values. (D) BRET validation of residues in the Gαq interface. See Table S7 for fitted parameter values where data represent mean ± SEM of n = 3 biological replicates. See also Table S5.

Figure 6.. HTR2A Couples Efficiently to Gq-Family Members In Vitro

(A)Shown is an alignment of the HTR2A-Gq (green and olive, respectively) and the M1-G11 (lavender and pink, respectively) interface at the tip of the α5 helix of the respective Gα subunits. As depicted, V359^H5.26 unde rgoes a 4.8 Å shift in HTR2A compared with M1 while Y356^H5.23 is shifted 2.9 Å relative to M1. (B) Shows a comparison of HTR2A-Gq with the HTR1B-Go (yellow and gray, respectively) showing that the HTR2A Y354^H5.26 cognate residue V246^H5.26 is shifted up 10.9 Å. (C) Shows a heatmap of the relative efficacy for selected agonists at HTR2A versus a reference agonist for 14 distinct Gα subunits. (D)Shows concentration-response curves (N = 3 biological replicates each) for the 14 Gα subunits in 7C along with controls for the Gz study. See also Table S7.

Figure 7.. Mutations of the ICL2 Residue I188 Differentially Modulate Gαq and Arrestin Interactions at HTR2A

(A) BRET2 HTR2A-Gαq assays reveal that I881A/E ^ICL2/34.51 mutations abolish 25CN-NBOH-potentiated Gαq activation; data represent mean ± SEM of n = 3 biological replicates. See Table S5 for fitted parameters. (B) BRET2 HTR2A-Gαq assays reveal that I881A/E ^ICL2/34.51 mutations abolished LSD-potentiated Gαq activation; data represent mean ± SEM of n = 3 biological replicates. See Table S5 for fitted parameters. (C) BRET2 HTR2A-Gαq assays reveal that I881A/E ^ICL2/34.51 mutations attenuate 5-HT-potentiated Gαq activation; data represent mean ± SEM of n = 3 biological replicates. See Table S5 for fitted parameters. (D–F) BRET1 HTR2A-βArr2 translocation assays show that I881A/E ^ICL2,34.51 mutations enhance the efficacy of 25CN-NBOH (D), LSD (E), and 5-HT (F); data represent mean ± SEM of n = 3 biological replicates. See Table S5 for fitted parameters. (G) The structural comparison in TM7 of active- (HTR2A-Gq, green and olive color, respectively) and inactive- (pink color) structures of HTR2A. (H) The structural comparison in ICL2 of active- (HTR2A-Gq, green and olive color, respectively) and inactive- (pink color) structures of HTR2A. (I) The structural comparison in ICL2^34.51 residue of β2AR-Gs (light green and gray, respectively, PDB: 3SN6) and β1AR-βarr1 (brown and lime, respectively, PDB: 6TKO). (J) The structural comparison in ICL2^34.51 residue of NT1- βarr1 (light blue and sky blue color, respectively, PDB: 6UP7) and M2R-βarr1 (magenta and yellow, respectively, PDB: 6U1N). (K) A diagram showing how ICL2E^34.51 differentially affects HTR2A-mediated G protein signaling and arrestin translocation.