This crystallography study analyzed the structure of LSD bound to a serotonin receptor and found that a branch of the receptor folds over the molecule while it is lodged into the binding pocket, and this lid-like structure secures LSD in place. This contributes to a slow dissociation rate of LSD, which forms the basis for its long-lasting effect. The authors suggest ways of introducing molecular mutations to selectively alter receptor signaling by increasing the mobility of this lid structure.
Introduction
The prototypical hallucinogen LSD acts via serotonin receptors, and ...
Methods
… here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B.
Results
The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD’s key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT2BR and 5-HT2AR-a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD’s slow binding kinetics may be due to a “lid” formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD’s binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment.
Discussion
This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD’s actions at human serotonin receptors.
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Lysergic acid diethylamide (LSD) is a highly potent psychoactive ergoline that produces prolonged changes in perception and mood and has historically informed both clinical and basic research into consciousness. Earlier work established that LSD interacts with many aminergic G-protein-coupled receptors (GPCRs), and that its psychedelic effects are primarily mediated via 5-HT2-family serotonin receptors, particularly 5-HT2A. Beyond canonical G-protein signalling, more recent studies have shown that LSD strongly engages b-arrestin pathways at biogenic amine GPCRs, a phenomenon of ‘‘biased agonism’’ whereby ligands stabilise receptor conformations that favour particular intracellular signalling cascades. The structural determinants that underlie LSD's signalling bias, potency and unusually long apparent duration of action remained unclear. Wacker and colleagues set out to clarify the molecular mechanisms of LSD action at serotonin receptors. Their aims were to (1) determine the crystal structure of LSD bound to human 5-HT2B receptor as a model for 5-HT2A, (2) provide a detailed functional characterisation of LSD's biased signalling profile, and (3) use the 5-HT2B/LSD structure to infer structural features relevant to LSD's activity at the homologous 5-HT2A receptor, the major in vivo target for psychedelic effects. The work combined X-ray crystallography, mutagenesis and functional assays, molecular dynamics (MD) simulations, and homology modelling/docking to link ligand chemistry, receptor conformation, binding kinetics and functional selectivity.
The core structural work used an engineered human 5-HT2B receptor construct expressed in Spodoptera frugiperda (Sf9) insect cells. The construct was modified (N- and C-terminal truncations, a thermostabilising M144W mutation, insertion of a thermostabilised apocytochrome b562 RIL (BRIL) in ICL3, and affinity tags) to enable purification and crystallisation. Receptor–LSD complexes were purified in detergent, reconstituted into lipidic cubic phase, and crystallised; X-ray diffraction data were collected at the Advanced Photon Source and the 5-HT2B/LSD complex structure was solved to 2.9 Å resolution by molecular replacement and refined with standard crystallographic software. Functional characterisation comprised several cell-based assays. Radioligand association/dissociation experiments used [3H]-LSD on membrane preparations to measure kinetic parameters (kon, koff) and residence times at 5-HT2B and 5-HT2A receptors, including engineered mutants (notably L209A in 5-HT2B and L229A in 5-HT2A). Gq-mediated signalling was assessed by calcium flux (Fluo-4/FLIPR) and phosphoinositide (IP) accumulation (scintillation proximity assay), while b-arrestin2 recruitment was measured with Tango luciferase-based assays and with a BRET assay utilising RLuc8-tagged receptor and Venus-tagged b-arrestin2. Time-course versions of these assays were used to probe the kinetics of signalling. Computational approaches included extensive MD simulations of the receptor in multiple conditions (LSD-bound, unliganded, L209A mutant, and ERG-bound template), performed with CHARMM36 force fields and Amber software on GPU hardware. The authors report over 100 ms of simulation across trajectories initiated from the crystal structures; production segments per trajectory ranged from 1.1–6.7 ms as reported. Analyses quantified ligand-pocket conformational changes and lid mobility (RMSF). A homology model of human 5-HT2A was built using the 5-HT2B/LSD structure as template and evaluated by ligand enrichment metrics; docking of LSD and constrained diethylamide analogues (SSAz, RRAz, LSA) was performed with DOCK3.7 to compare poses and predict interactions. Mutagenesis studies (L209A/L229A) were used to test structure-derived hypotheses about a putative extracellular loop 2 (EL2) ‘‘lid’’ and its effect on ligand kinetics and signalling bias.
The researchers solved the 5-HT2B receptor crystal structure bound to LSD at 2.9 Å. In the structure LSD occupies the orthosteric pocket and extends into a previously described extended binding site; a conserved salt bridge links the basic nitrogen of LSD to D1353.32 (helix III). The ergoline (tryptamine-like) core sits in a narrow hydrophobic cleft, forming aromatic contacts with phenylalanines in helix VI and a hydrogen bond to the backbone of G2215.42 in helix V. LSD's diethylamide substituent occupies a crevice between helices II, III and VII, with one ethyl contacting L1323.29/W1313.28 and the other projecting toward L3627.35. Comparison with the ergotamine (ERG)-bound 5-HT2B structure revealed a distinct binding pose for LSD: the ergoline core of LSD is located higher (shallower) in the pocket, nearer extracellular loop 2 (EL2), whereas ERG sits deeper and hydrogen bonds to T1403.37. Multiple side-chain rotamer changes (for example T1142.64, E3637.36, M2185.39) and larger helical shifts accompany the different ligands. Computational MD simulations supported ligand-dependent conformational preferences: simulations initiated from ligand-removed structures showed M2185.39 adopting an ‘‘up’’ conformation matching the LSD-bound structure, and the authors report having performed over 100 ms of simulation overall. Quantitatively, calculated pocket volumes decreased from 2898.7 to 2068.4 Å3 (a 28.6% reduction) between the ERG- and LSD-bound structures in one analysis. To probe the functional relevance of the diethylamide conformation, the team tested sterically constrained LSD analogues. The (S,S)-azetidide (SSAz), whose constrained diethylamide resembles LSD's receptor-bound conformation, showed nearly identical potency and efficacy to LSD for b-arrestin2 recruitment at both 5-HT2B and 5-HT2A receptors. In contrast, the (R,R)-azetidide (RRAz) and lysergamide (LSA) displayed much reduced potencies for b-arrestin2 recruitment; differences in Gq-mediated calcium flux were smaller. Docking into a 5-HT2A homology model recapitulated the LSD pose and predicted that SSAz adopts a similar orientation to LSD, whereas RRAz and LSA orient their amide substituents away from hydrophobic receptor contacts. Kinetic binding experiments showed very slow dissociation of [3H]-LSD: at 25 °C the 5-HT2B dissociation half-life (t1/2) exceeded 5 hr, and at 37 °C the residence time at 5-HT2B was approximately 46 min (koff = 0.022 ± 0.004 min-1). The LSD-bound structure reveals residues 207–214 of EL2 forming a ‘‘lid’’ over the ligand that likely hinders egress. MD indicated that EL2 occasionally adopts more open conformations, and that L209 in EL2 makes extensive hydrophobic contacts with LSD and transmembrane residues. Mutation of this latch residue to alanine (L209A in 5-HT2B) increased lid mobility in simulations (root-mean-square fluctuation differences significant at p < 0.01) and decreased LSD residence time by roughly 10-fold (from 44 min to 4.3 min at 37 °C in the reported assays), while accelerating the apparent on-rate and leaving steady-state affinity largely unchanged. ERG binding kinetics were minimally affected by the L209A mutation. Functionally, the L209A mutant selectively attenuated LSD-mediated b-arrestin2 recruitment potency and efficacy without substantially altering Gq-mediated calcium flux or IP accumulation. Parallel findings were obtained for 5-HT2A: docked LSD in the 5-HT2A model made contacts with EL2 residue L229 (analogous to L209), and [3H]-LSD dissociation from wild-type 5-HT2A was even slower (koff = 0.005 ± 0.001 min-1; residence time ≈221 min), whereas the L229A mutation reduced residence time to ≈50 min and selectively reduced arrestin recruitment. Time-course BRET and IP accumulation assays showed that both b-arrestin2 recruitment and Gq signalling increased with prolonged compound incubation for wild-type receptors, consistent with the prolonged residence times; however, the EL2 alanine mutants failed to show the time-dependent augmentation of b-arrestin2 recruitment while time-dependency of Gq signalling was largely preserved. Transduction coefficients (log(t/KA)) computed over time revealed a time-dependent increase for both pathways in wild-type receptors, and selective abrogation of the arrestin time-dependence in L209A/L229A mutants.
Wacker and colleagues interpret their data as providing the first structure-informed explanation for key aspects of LSD pharmacology. They highlight two principal conclusions. First, the diethylamide substituent of LSD adopts a constrained conformation within the receptor binding site that is critical for potency and for the stereochemical preferences observed with close analogues; constrained analogues that mimic this conformation (SSAz) reproduce LSD's arrestin-biased functional properties, whereas stereoisomers that cannot adopt it (RRAz, LSA) are less active. Second, interactions between LSD and an EL2 ‘‘lid’’—notably contacts with L209 in 5-HT2B (and the corresponding L229 in 5-HT2A)—appear to underlie LSD's unusually slow dissociation kinetics and its time-dependent promotion of b-arrestin translocation. The authors propose that the EL2-derived lid can function as a latch, limiting ligand egress and thereby prolonging receptor occupancy; experimentally accelerating LSD kinetics by L209A/L229A mutations selectively reduced arrestin recruitment without markedly affecting Gq signalling. They place these findings in context by noting that slow residence time has long been observed for LSD in crude membrane studies, but the 5-HT2B/LSD structure and supporting MD provide a molecular mechanism linking ligand chemistry to receptor conformational dynamics and biased signalling. The researchers acknowledge limitations: structural snapshots and simulations cannot fully account for complex central nervous system effects or definitively predict in vivo efficacy, and the timescales of simulations are far shorter than ligand dissociation in vitro. They also note that LSD's plasma clearance (reported t1/2 ≈3.6 hr) complicates simple correlations between receptor residence time and behavioural duration. Finally, the authors suggest implications for future work: the structure may enable structure-based discovery of novel chemotypes at 5-HT2A and 5-HT2B that could disentangle hallucinogenic effects from other therapeutic activities of 5-HT2A agonists, and their results provide mechanistic hypotheses to test in further pharmacological and in vivo studies.
Generation of 5-HT 2B R receptor crystallization construct Crystallization of the 5-HT 2B R/LSD complex was done based on a previously engineered receptor construct that was edited by Quickchange PCR. Using site-directed mutagenesis we added the ICL3 residue V313 to a previously published constructthat had been synthesized by DNA2.0. The final construct a) lacks N-terminal residues 1-35, b) lacks C-terminal residues 406-481, c) contains a thermostabilizing M144W 3.41 mutation, and d) contains A1-L106 of the thermostabilized apocytochrome b 562 RIL (BRIL) from E. coli (M7W, H102I, R106L) in place of receptor residues Y249-S312 of ICL3. Further modifications are a haemagglutinin (HA) signal sequence followed by a FLAG tag at the N terminus, and a PreScission protease site followed by a 10 3 His tag at the C terminus to enable purification by immobilized metal affinity chromatography. Expression and purification of 5-HT 2B R High-titer recombinant baculovirus (> 10 9 viral particles per ml) was generated using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Recombinant baculovirus was obtained by transfecting $5 mg of recombinant bacmid into 5x10 5 settled Spodoptera frugiperda (Sf9) cells (Expression Systems) in a 24 well plate (Corning) using 3 ml Cellfectin II Reagent (Invitrogen). After 5-12 hr, media was exchanged for 1 mL Sf-900 II SFM media (Invitrogen) and incubated for 4-6 days at 27 C. P0 viral stock with $10 9 virus particles per ml was harvested as the supernatant and used to generate high-titer baculovirus stock by infection of 40-1000 mls of Sf9 cells and incubation for several days. Viral titers were determined by flow-cytometric analysis of cells stained with gp64-PE antibody (Expression Systems). Expression of 5-HT 2B R was carried out by infection of Sf9 cells at a cell density of 2-3 3 10 6 cells/ml in ESF921 media (Expression Systems) with P1 or P2 virus at a MOI (multiplicity of infection) of 3-5. Cells were harvested by centrifugation at 48 hr post infection, washed in PBS, and stored at À80 C until use. Cells were disrupted by thawing frozen cell pellets in a hypotonic buffer containing 10 mM HEPES, pH 7.5, 10 mM MgCl 2 , 20 mM KCl and protease inhibitors (500 mM AEBSF, 1 mM E-64, 1 mM Leupeptin, 150 nM Aprotinin). Membranes were purified by repeated centrifugation in a high osmolarity buffer containing 1.0 M NaCl, 10 mM HEPES, pH 7.5, 10 mM MgCl 2 , 20 mM KCl, to remove soluble and membrane associated proteins. Purified membranes were directly flash-frozen in liquid nitrogen and stored at À80 C. Purified membranes were resuspended in buffer containing 10 mM HEPES, pH 7.5, 10 mM MgCl 2 , 20 mM KCl, 150 mM NaCl, 50 mM LSD (synthesized in house), and protease inhibitors, and incubated at room temperature for 1 hr. After 30 min incubation in the presence of 2 mg/ml iodoacetamide (Sigma), membranes were solubilized in 10 mM HEPES, pH 7.5, 150 mM NaCl, 1% (w/v) n-dodecyl-b-D-maltopyranoside (DDM, Anatrace), 0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma), 25 mM LSD, and protease inhibitors for 2 hr at 4 C. Unsolubilized material was removed by centrifugation at 150,000 3 g for 30 min, and 15 mM imidazole was added to the supernatant. Proteins were bound to TALON IMAC resin (Clontech) overnight at 4 C using approximately 750 ml resin for protein purified from 1 L of cells. The resin was then washed with 10 column volumes (cv) of Wash Buffer I (50 mM HEPES, pH 7.5, 800 mM NaCl, 0.1% (w/v) DDM, 0.02% (w/v) CHS, 20 mM imidazole, 10% (v/v) glycerol, and 20 mM LSD, followed by 10 cv of Wash Buffer II (25 mM HEPES, pH 7.5, 500 mM NaCl, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 10% (v/v) glycerol, and 20 mM LSD). Proteins were eluted in 2.5 cv of Wash Buffer II + 250 mM imidazole, concentrated in a 100 kDa molecular weight cut-off Vivaspin 20 concentrator (Sartorius Stedim) to 500 ml, and imidazole was removed by desalting the protein over PD MiniTrap G-25 columns (GE Healthcare). The C-terminal 10 3 His-tag was removed by addition of His-tagged PreScission protease (GenScript) and incubation overnight at 4 C. Protease, cleaved His-tag and uncleaved protein were removed by passing the suspension through equilibrated TALON IMAC resin (Clontech) and collecting the flow-through. 5-HT 2B R/LSD complexes were then concentrated to $40 mg/ml with a 100 kDa molecular weight cut-off Vivaspin 500 centrifuge concentrator (Sartorius Stedim). Protein purity and monodispersity were tested by analytical size-exclusion chromatography.
A molecular understanding of the structural basis of psychoactive drug action has long been elusive. LSD, with its profound activity on human perception and awareness, is one of the most prominent psychoactive drugs. Whereas it has long been thought that LSD and many other hallucinogens act at serotonin receptors, understanding LSD's actions at a molecular level has remained clouded, notwithstanding important computational. The structure of LSD bound to one of its molecular targets, and the signaling and simulation studies it enables, begins to address long-standing questions about the relation of LSD's chemical structure to its activity, kinetics, and signaling. Two noteworthy observations stand out. First, the key amide side chain of LSD-the group that distinguishes it from the far less hallucinogenic lysergamide (LSA)-adopts a constrained conformation in the binding site that cannot exchange readily with alternative conformational states. This conformation, and by extension the contacts made, is crucial for LSD's actions, and close analogs See also Figureand Tablesand. that cannot adopt it are much less active in vivo. Second, this conformation apparently contributes to LSD's relatively potent ability to promote b-arrestin translocation. The structure of the 5-HT 2B R/LSD complex reveals that the amide substituents, such as LSD's diethylamide, largely determine the positioning of the ergoline system within the orthosteric pocket. This new structure explains the previously enigmatic requirement of LSD and related lysergamides for a specific conformation of the diethylamide substituents for activity. The observation that, for instance, the probe molecule SSAz is active, whereas its stereoisomer RRAz is less activewas difficult to reconcile with the prior small molecule crystal structure of LSD alone, in which the diethylamide adopts a different conformation. As the receptor-LSD complex structure shows, the diethylamide of receptor-bound LSD adopts a conformation consistent with the observed stereochemical preference for SSAz over RRAz at both the 5-HT 2A and 5-HT 2B receptors. The diethylamide positioning and interactions could also contribute to LSD's long residence time at 5-HT 2B R and 5-HT 2A R-its presumed major molecular target. MD simulations suggest the slow kinetics of LSD are due, at least in part, to a lid formed by EL2 covering the binding pocket. Compellingly, accelerating LSD's binding kinetics by making a substitution to a key residue identified structurally (L209 EL2 ), selectively attenuates the time-dependent augmentation of b-arrestin2 recruitment while minimally affecting Gq signaling. We note in this regard that, although structural studies cannot provide definitive insights into drug actions in vivo, it is conceivable that LSD's long residence time via EL2 interactions could contribute to LSD's long duration of action, despite its apparent rapid clearance from the body [t 1/2 = 3.6 hr]. Crystal structures and molecular simulations can never fully explain CNS drug efficacy, which for LSD requires integrative action over complex neural networks, leading to highly distinctive cognitive effects. Our observations nevertheless provide the first structure-informed insights into the molecular actions for any hallucinogen. Our findings explain the role of LSD functional groups whose importance in vivo has long been recognized, but whose mechanism has been opaque. The structure-informed insights also link these particular interactions to the unusual signaling kinetics of LSD-particularly as it relates to b-arrestin translocation, effects that could be crucial for its hallucinogenic activity in vivo (W.C. Wetsel, R.M. Rodriguez, and B.L.R., unpublished data). Finally, this structure may template future structure-based efforts to discover new chemotypes at 5-HT 2A and 5-HT 2B receptors. Such molecules could help disentangle hallucinogenic effects from other intriguing activities of 5-HT 2A agonists, something that has heretofore been impossible but that a structure-based approach, with its ability to identify novel chemotypes, now allows.
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