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

This study reveals structurally how psychedelics, including LSD, psilocin, mescaline, and various N-BOH analogs, mediate their therapeutic and hallucinogenic effects by binding to and activating their molecular target, the serotonin (5-HT) 2A receptor coupled with G-protein Gaq.

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.”

Authors: Kuglae Kim, Tao Che, Ouliana Panova, Jeffrey F. DiBerto, Jiankun Lyu, Brian E. Krumm, Daniel Wacker, Michael J. Robertson, Alpay B. Seven, David E. Nichols, Brian K. Shoichet, Georgios Skiniotis & Bryan L. Roth

Notes

This paper is included in our ‘Top 10 Articles on Psychedelics in the Year 2020‘

This paper was accompanied by an explanatory article in EurkAlert, and reported on in Psychology Today and Inverse.

From the last one ““Now we know how psychedelic drugs work – finally!” he tells Inverse. “Now we can use this information to, hopefully, discover better medications for many psychiatric diseases.””

Summary

INTRODUCTION

Naturally occurring psychedelics from plants have been used for millennia for religious purposes and shamanism. More recently, so-called ”designer hallucinogens” with a scaffold related to N-benzyl-2,5-dimethoxy-phenethylamine have become popular, albeit with scattered reports of toxicity.

Hallucinogens like psilocybin and LSD have been reported to have potential therapeutic actions for many neuropsychiatric diseases, including depression and anxiety.

The active-state structure of 5-HT2A bound to 25CN-NBOH in complex with a mini-Gaq-bg heterotrimer stabilized by a single-chain variable fragment (scFv16) was obtained by cryoelectron microscopy. This structure provides insights into how a model hallucinogen stabilizes a specific HTR2A transducer-coupled state.

AGaq-Coupled HTR2A Structure Bound to the Hallucinogen 25CN-NBOH

We engineered a mini-Gaq protein to faithfully recapitulate wild-type Gaq-coupled G-protein-coupled receptor activation for several GPCRs, including HTR2A. Bioluminescence resonance energy transfer (BRET) studies revealed that the functional activity of this engineered mini-Gaq construct is comparable to the wild-type Gaq.

To improve the expression of HTR2A, parts of the N- and C-terminal were truncated, and a pFAST dual expression vector was used to express HTR2A and the GaqiN-Gb1-Gg2 heterotrimer. Single-particle cryo-EM of HTR2A-Gaq/b1/g2 complexed with 25CN-NBOH was determined at a global nominal resolution of 3.27 A , enabling near-atomic resolution modeling of the complex. The overall structure of the complex is consistent with the fully active-state receptor conformation.

Structures of LSD- and Methiothepin-Bound HTR2A

To obtain a more thorough understanding of how hallucinogens might interact with HTR2A, we determined the crystal structures of two HTR2A-ligand complexes: the prototypical hallucinogen LSD and the potent inverse agonist methiothepin. The structures show that the orientation of the receptors is nearly identical, although slight differences are observed.

The electron density maps for LSD and methiothepin were well resolved, and the structure of the LSD-HTR2A complex is similar to the structure of the LSD-HTR2B complex. Methiothepin is a potent and efficacious HTR2A inverse agonist.

Structural Comparisons of HTR2A Inactive- and ActiveState Structures

We first assessed the conformational rearrangements between the inactive and active state structures, and found that the Gaq-coupled state is considerably more open than the LSD-bound state. The solvent-accessible volumes of the ligand-binding pockets were 188.1 A3 for methiothepin, 153.1 A3 for LSD, and 287.0 A3 for 25CN-NBOH.

The cryo-EM map shows that the transmembrane domains and the G protein binding interface are well resolved, and that the intracellular ends of TM5 and TM6 are tilted outward by 4.3 A and 7.1 A, respectively, upon agonist binding and transducer coupling.

The structure of HTR2A is stabilized by Gaq coupling, and ICL2 displays helical turns reminiscent of that predicted by computational studies. Several conserved motifs are altered during activation, but the initiation and sequence of these events are still understudied.

25CN-NBOH Displays a Distinct Binding Mode at HTR2A

We next examined the ligand-binding pocket of the three structures, which revealed a shared mechanism for HTR2A binding. A salt bridge was observed between D1553.32 and a positively charged nitrogen in each ligand, which is a critical interaction for ligand binding in serotonin and other monoamine receptors.

HTR2A-selective agonist 25CN-NBOH displays a unique binding pose compared to non-selective partial agonist LSD and non-selective inverse agonists methiothepin, risperidone, and zotepine. This binding pose is thought to be related to the pivotal role of W3366.48 in controlling GPCR signaling transduction.

The P5.50-I3.40-F6.44 motif of the serotonin-receptor HTR2A undergoes conformational changes upon receptor activation, which may partially explain how allosteric changes are transmitted in the active state similar to what was observed in the CB1 cannabinoid-Gi in complex with fubinaca. 25-CN-NBOH forms a hydrogen bond with S1593.36, and the 2-hydroxyphenyl moiety is accommodated by the conserved G3697.42, which moves ”in” upon agonist binding to form hydrophobic interactions. The binding affinity of G369A7.42 does not significantly change compared to the wild-type.

25CN-NBOH binds to HTR2A and has negligible affinity for all other biogenic amine receptors, while preferring HTR2A over HTR2C and HTR2B. This could contribute to 25CN-NBOH’s selectivity at 5HTR2A over many other biogenic amine receptors.

25CN-NBOH’s phenethylamine group is located in the orthosteric pocket similar to LSD and other antagonists, but 25CN-NBOH does not have substantial interactions with the conserved serines S2395.43 and S2425.46. This unique residue may contribute to LSD’s unusually long binding kinetics.

We used molecular docking to evaluate the potential binding modes of 5-HT and found that the positively charged primary amine forms a saltbridge with the anionic D1553.32 and is stabilized by aromatic interactions with F3406.52 and F3396.51 and hydrogen bonds with N3436.55 and S2425.46.

The Agonist-Gaq Complex Binding Interface

The C-terminal helix (a5 helix) of Gaq forms a major interface with HTR2A, and several residues on HTR2A form H-bonds with Gaq. Additionally, several residues on HTR2A form a hydrophobic core with Gaq.

HTR2A and Gaq proteins maintain robust whole cell and receptor cell surface expression, although some mutations slightly decreased receptor expression levels compared to wild-type. Q237AH5.17 and N244AH5.24 are conserved residues in the Gaq family that may be involved in Gaq specificity.

We compared the Gq interface with the recent M1-muscarinic G11 interface and found that the terminal hydroxyl of Y243 was shifted 2.9 A to create additional interactions with HTR2A, and the terminal N-L-V motif was displaced downward to achieve a previously unreported interacting surface with TM7 of M1.

Recent studies with chimeric G proteins have indicated that GPCRs may interact with a much larger diversity of Ga subunits than previously anticipated. The 5-HT2A-Gq structure reveals non-conserved interactions with several members of the Gi family and minimally with Gas family members.

We tested whether HTR2A would interact with Gao or related Gai proteins using our recently described BRET-based technology. Our results showed that HTR2A coupled robustly to Gq-family members and minimally to conventional Gi- or Gs family members.

Gaq is activated by I181(ICL2)34.51, which interacts with L34S1.02, V79S3.01, F228H5.08, and I235H5.15 via hydrophobic interactions. We found that I181A34.51 and I181E34.51 mutations attenuated or abolished Gaq activation by 25CN-NBOH and 5-HT respectively.

In Family A aminergic receptors, the phenolic hydroxyl group of Y5.58 is located in the inner core of TM6, whereas in HTR2A, HTR2B, and HTR2C, the phenolic hydroxyl group of Y5.58 is displaced outward from TM6.

DISCUSSION

In this paper, we determined the structure of the 5-HT2A serotonin receptor coupled to its canonical transducer Gaq. We also identified key determinants essential for agonist actions and receptor-Gq coupling.

Some of the findings contrast with predictions from a recent study utilizing chimeric Ga subunits that indicated that HTR2A interact efficiently with all of the 11 tested Ga subunits, albeit with low efficacy at Gas. However, using full-length heterotrimeric G proteins, we find that HTR2A couples weakly with only one Gi-family member, Gaz.

We found that a key hydrophobic residue essential for G protein coupling among various GPCRs – I181ICL2 – when mutated, abolishes Gaq coupling while potentiating HTR2A-Gq interactions.

A 3.3 A resolution structure of the turkey b1-adrenergic receptor complexed with human b-arrestin1 was reported. This structure provides a potential structural explanation for how loss of this hydrophobic interaction impairs Gq subunit coupling while preserving arrestin interaction – at least for HTR2A.

Our findings provide fundamental insights into GPCR-Gq interactions and have relevance for neuropsychiatric drug discovery. These studies will provide a framework for a structure-guided search for more selective and efficacious HTR2A agonists as potential innovative neuropsychiatric therapeutics.

STAR+METHODS

The experimental model and subject details are as follows: generation of HTR2A constructs, expression, purification, formation of heterotrimeric mini-Gaq protein complex, cryoEM data collection, 3D reconstruction, model building, and refinement, lipidic cubic phase crystallization, and structure determination.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

To crystallize the 5-HT2A receptor, a modified thermostabilized apocytochrome b562RIL (BRIL) was inserted into the receptor’s third intracellular loop (ICL3) at A265 and T311 of the human 5-HT2A gene. This receptor was further optimized by truncation of N-terminal residues 1-65 and C-terminal residues 405-471.

Purification for HTR2A-XTAL and -cryoEM

Thawed insect cell membranes were disrupted in a hypotonic buffer containing 10 mM HEPES (pH 7.5), 10 mM MgCl2, and 20 mM KCl and protease inhibitors. The membranes were then purified and incubated with desired ligands and protease inhibitor cocktail. The solubilized HTR2A proteins were isolated by ultra-centrifugation and then incubated with TALON IMAC resin, 800 mM NaCl and 20 mM imidazole. They were washed with 10 column volumes of washing buffer I and II, without imidazole. Protein was eluted from a gel using 3 column volumes of elution buffer and concentrated in a Vivaspin 20 concentrator. The 5HT2A protein was purified using PD MiniTrap G-25 columns, His-tagged PreScission protease, TALON IMAC resin and a Vivaspin 500 centrifuge concentrator. The protein purity and monodispersity were tested by analytical size-exclusion chromatography column, SRT-300. For the CryoEM construct purification, 500 mL of 5HT2A protein sample was applied to PD MiniTrap G-25 columns (GE Healthcare), and the N-terminal BRIL was removed by addition of His-tagged PreScission protease (GeneScript). The flow-through was collected until use.

Model building and refinement

Homology models of active-state HTR2A were built using SWISS-MODEL and docked into the EM density map using Chimera. The model statistics was validated using Molprobity.

Radioligand binding assays

Competitive binding assays were performed using membrane preparations from HEK293 T cells transiently expressing HTR2A wt or mutants. The binding assays were set up in 96-well plates in the standard binding buffer and the results were analyzed using the equation ‘one-site fit Ki’ in GraphPad Prism 8.0.

Ligand Dissociation Radionligand Binding Assay

Radioligand dissociation assays were performed in parallel with competitive binding assays using the same concentrations of radioligand, membrane preparations, and binding buffer. Time points spanned 2 minutes to 7 hours and were harvested by vacuum filtration onto 0.3% polyethyleneimine pre-soaked 96-well filter mats.

Bioluminescence resonance energy transfer assays (BRET)

HEK293T cells were co-transfected with human 5-HT2AR containing C-terminal Renilla luciferase (RLuc8) and Venus-tagged N-terminal b-arrestin2 and plated in poly-lysine coated 96-well white clear bottom cell culture plates. The cells were incubated at 37 C for 20 minutes before receiving drug stimulation. The net BRET ratio was calculated as a function of drug concentration using Graphpad Prism 5 and analyzed using nonlinear regression.

HEK293T cells were transfected with HTR2A-mediated G protein activation DNA and harvested the next day. The cells were plated in poly-D-lysine-coated 96-well white assay plates.

After plating in 96-well assay plates, cells were treated with 50 mM coelenterazine 400a in 60 mL of drug buffer for an additional 5 minutes. BRET ratio was computed as the ratio of the GFP2 emission to rLuc8 emission, and data were normalized to % 5-HT stimulation.

Surface expression enzyme-linked immunosorbent assay

To confirm cell surface expression of HTR2A and its mutants, cells were transfected for 48 hours and then stained with a monoclonal ANTI-FLAG M2-Peroxidase (HRP) antibody. Then, a SuperSignal enzyme-linked immunosorbent assay (ELISA) Pico Chemiluminescent Substrate was added and luminescence was counted.

QUANTIFICATION AND STATISTICAL ANALYSIS

Data were analyzed using Graphpad Prism 8.0 for the radioligand binding assays and the equation ‘one-site fit Ki’ in Graphpad Prism 8.0 for the inhibition studies. The data were normalized to % 5-HT or the reference agonist stimulation and analyzed using nonlinear regression ”log(agonist) vs. response” in GraphPad Prism 8.0.