To obtain structural insights into LSD’s actions at human serotonin receptors, we crystallized an engineered 5-HTR construct bound to LSD by extensively modifying our previous approach (). We eventually obtained crystals and solved the X-ray structure of the 5-HTR/LSD complex to a resolution of 2.9 Å ( Table 1 Figures 1 and S1 ). LSD is bound in the orthosteric binding site while also engaging the previously described extended binding site of the receptor ( Figures 1 A–1C) (). As an ergoline, LSD’s tryptamine moiety, which resembles that of 5-HT, is embedded in a tetracyclic scaffold ( Figure 1 D). Ergolines exhibit diverse amide modifications, such as LSD’s diethylamide that is essential for its optimal potency in vivo (), or the peptide moiety of ergotamine (ERG) ( Figure 1 D). LSD is anchored to 5-HTR by a conserved salt bridge between D135in helix III and the basic nitrogen of the ergoline system ( Figures 1 B–1D), an interaction that has been observed consistently in aminergic receptor structures (). The ergoline system of LSD occupies the orthosteric pocket, which forms a narrow cleft lined mainly by hydrophobic side chains from residues in helices III, V, VI, and VII; such a cleft is common to most biogenic amine receptors. LSD’s ergoline ring system forms edge-to-face aromatic contacts with conserved phenylalanines (F340, F341) in helix VI, as previously anticipated for its complex with 5-HTR (), and hydrogen bonds with the backbone of G221in helix V. LSD’s diethylamide group binds in a crevice between helices II, III, and VII, where one ethyl group forms non-polar contacts with L132and W131, while the other ethyl group extends toward L362—residues previously shown to be part of an extended binding pocket in 5-HTand 5-HTreceptors () ( Figure 1 C).

(A) Structure of LSD (magenta) bound 5-HT 2B R (lightblue) with 2Fo-Fc electron density map of LSD (blue mesh) contoured at 1σ. (B) Structure of LSD bound 5-HT 2B R with Fo-Fc omit electron density map of LSD (green mesh) contoured at 3σ. (C) 2Fo-Fc electron density map of 5-HT 2B R binding pocket residues (blue mesh) contoured at 1σ.

(D) 2D representation of LSD, Ergotamine (ERG), and 5-hydroxytryptamine (5-HT/serotonin). LSD belongs to the class of ergolines like ERG and contains a diethylamide substituent (highlighted in light blue) connected to the ergoline scaffold (highlighted in yellow). Ergolines contain a tryptamine core scaffold (dark blue) like the endogenous ligand 5-hydroxytryptamine (5-HT/serotonin). Diagram of interactions between LSD and the receptor in the ligand binding pocket is shown, with the hydrogen bonds between D135 3.32 and the LSD basic nitrogen in yellow, and G221 5.42 and the LSD indole nitrogen indicated by red dashes, respectively. Residues are labeled according to Ballesteros-Weinstein nomenclature. Residues highlighted in red show significant changes between LSD- and ERG-occupied 5-HT 2B R, while residues highlighted in green show a significant interaction with ERG but not LSD.

(A) 5-HT 2B R cartoon representation (light blue) with helices labeled according to GPCR nomenclature. LSD is shown as a stick model with carbons, nitrogens, and oxygens colored in magenta, blue, and red, respectively. The LSD stick model is overlaid with a semi-transparent surface representation of the compound.

LSD’s Distinct Binding Pose

Compared to ERG, the ergoline moiety of LSD is located higher in the orthosteric pocket, closer to EL2 and the extracellular space, adopting a shallow binding mode. ERG is located deeper in the pocket with its indole nitrogen hydrogen bonding to T1403.37 in helix III, at the bottom of the pocket, further embedded in the intra-membrane region. In contrast, the indole nitrogen of LSD does not interact with T1403.37 in helix III, but instead hydrogen bonds with the backbone oxygen of G2215.42 in helix V.

2B R: T1142.64, E3637.36, and M2185.39 all change their rotamer states between the two structures ( 2B R/ERG complex, the phenyl moiety of ERG appears to “push” down on M2185.39, wedging the M2185.39 side chain between the peptide and ergoline moiety of ERG ( 2B R/LSD complex, by contrast, the diethylamide ergoline substituent does not interact with M2185.39. As a result the M2185.39 side chain flips up, allowing more space for LSD to adopt a shallower binding mode. We also examined these differential binding modes by molecular dynamics (MD) simulations, which provided additional support for the hypothesis that the binding of LSD preserves the unliganded conformation of M2185.39, whereas binding of ERG distorts it. In MD simulations initiated from structures of either the ERG-bound or LSD-bound 5-HT 2B R but with the ligand removed, the M2185.39 side chain consistently adopted an upward conformation matching that of the LSD-bound structure ( Figure S3 2B R Complexes, Differences in LSD versus Ergotamine Surfaces, and Conformational Changes between 5-HT 2B R/LSD and 5-HT 2B R/ERG Binding Pockets Compared with Activation-Related Changes Observed in the β 2 AR Binding Pocket, Related to MD Simulations of 5-HTR Complexes, Differences in LSD versus Ergotamine Surfaces, and Conformational Changes between 5-HTR/LSD and 5-HTR/ERG Binding Pockets Compared with Activation-Related Changes Observed in the βAR Binding Pocket, Related to Figures 2 and 4 Show full caption 5.39 matches its unliganded conformation, as M2185.39 adopts the conformation observed in the LSD-bound crystal structure in simulations initiated from both the ERG-bound and LSD-bound structures but with the ligand removed. The conformations shown for LSD-bound and ERG-bound 5-HT 2B R are from crystal structures. The conformation shown for unliganded 5-HT 2B R is from simulations. (B) Convergence analysis for MD simulations of L209AEL2 5-HT 2B R. Each trace represents the root mean square deviation (RMSD) of the EL2 lid from the LSD-bound crystal structure in a simulation of the LSD-bound L209AEL2 mutant. At the beginning of each simulation, the RMSD increases, indicating that the lid is relaxing away from its initial conformation, partly as a result of the mutation. However, this increase levels off within the first 300 ns of simulation. RMSD is computed over all non-hydrogen atoms of residues 207–214. Traces are smoothed by a 60-ns moving average. Note that EL2 continues to fluctuate even when its RMSD from the initial structure is no longer systematically increasing, and that this analysis does not demonstrate convergence in the strict sense that each simulation fully samples the Boltzmann distribution. We performed multiple simulations under each condition, and 2B R/LSD complex with LSD in magenta while (D) shows surface representation of 5-HT 2B R/ERG complex with ERG in green. (E) Superposition of ligand binding sites of 5-HT 2B R (light blue) bound to LSD (transparent magenta), and 5-HT 2B R (light green) bound to ERG (transparent dark green)(PDB ID: Wacker et al., 2013 Wacker D.

Wang C.

Katritch V.

Han G.W.

Huang X.P.

Vardy E.

McCorvy J.D.

Jiang Y.

Chu M.

Siu F.Y.

et al. Structural features for functional selectivity at serotonin receptors. 2 AR (purple) bound to the inverse agonist ICI 118,551 (transparent cyan) (PDB ID: Wacker et al., 2010 Wacker D.

Fenalti G.

Brown M.A.

Katritch V.

Abagyan R.

Cherezov V.

Stevens R.C. Conserved binding mode of human beta2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography. 2 AR (salmon) bound to the covalent agonist FAUC50 (transparent light pink)(PDB ID: Rosenbaum et al., 2011 Rosenbaum D.M.

Zhang C.

Lyons J.A.

Holl R.

Aragao D.

Arlow D.H.

Rasmussen S.G.

Choi H.J.

Devree B.T.

Sunahara R.K.

et al. Structure and function of an irreversible agonist-β(2) adrenoceptor complex. 2 AR (purple) bound to the inverse agonist ICI 118,551 (transparent cyan) (PDB ID: Wacker et al., 2010 Wacker D.

Fenalti G.

Brown M.A.

Katritch V.

Abagyan R.

Cherezov V.

Stevens R.C. Conserved binding mode of human beta2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography. 2 AR (dark yellow) bound to the agonist BI 167107 (transparent orange pink)(PDB ID: Rasmussen et al., 2011a Rasmussen S.G.

Choi H.J.

Fung J.J.

Pardon E.

Casarosa P.

Chae P.S.

Devree B.T.

Rosenbaum D.M.

Thian F.S.

Kobilka T.S.

et al. Structure of a nanobody-stabilized active state of the β(2) adrenoceptor. 2B /ERG versus 5-HT 2B /LSD the ligand-binding pocket RMSD is 0.99 Å while for the active versus inactive β 2 AR structures the RMSD is 0.85 Å. The RMSD values were computed over all non-hydrogen backbone and side-chain atoms in transmembrane helix residues that are in the union of (i) the LSD-binding pocket and the ERG-binding pocket for 5-HT 2B R and (ii) the ICI 118,551-binding pocket and the BI-binding pocket for β 2 AR (binding pocket residues are defined as those within 4.5 Å of the ligand in the crystal structure). (A) MD simulations suggest that the LSD-bound conformation of M218matches its unliganded conformation, as M218adopts the conformation observed in the LSD-bound crystal structure in simulations initiated from both the ERG-bound and LSD-bound structures but with the ligand removed. The conformations shown for LSD-bound and ERG-bound 5-HTR are from crystal structures. The conformation shown for unliganded 5-HTR is from simulations. (B) Convergence analysis for MD simulations of L209A5-HTR. Each trace represents the root mean square deviation (RMSD) of the EL2 lid from the LSD-bound crystal structure in a simulation of the LSD-bound L209Amutant. At the beginning of each simulation, the RMSD increases, indicating that the lid is relaxing away from its initial conformation, partly as a result of the mutation. However, this increase levels off within the first 300 ns of simulation. RMSD is computed over all non-hydrogen atoms of residues 207–214. Traces are smoothed by a 60-ns moving average. Note that EL2 continues to fluctuate even when its RMSD from the initial structure is no longer systematically increasing, and that this analysis does not demonstrate convergence in the strict sense that each simulation fully samples the Boltzmann distribution. We performed multiple simulations under each condition, and Figure 4 F shows aggregate statistics across these simulations. (C) Shows surface representation of 5-HTR/LSD complex with LSD in magenta while (D) shows surface representation of 5-HTR/ERG complex with ERG in green. (E) Superposition of ligand binding sites of 5-HTR (light blue) bound to LSD (transparent magenta), and 5-HTR (light green) bound to ERG (transparent dark green)(PDB ID: 4IB4 )). (F) Superposition of ligand binding sites of the inactive state βAR (purple) bound to the inverse agonist ICI 118,551 (transparent cyan) (PDB ID: 3NY8 ), and inactive state βAR (salmon) bound to the covalent agonist FAUC50 (transparent light pink)(PDB ID: 3PDS )). (G) Superposition of ligand binding sites of the inactive state βAR (purple) bound to the inverse agonist ICI 118,551 (transparent cyan) (PDB ID: 3NY8 ), and the active state βAR (dark yellow) bound to the agonist BI 167107 (transparent orange pink)(PDB ID: 3P0G )). For the 5-HT/ERG versus 5-HT/LSD the ligand-binding pocket RMSD is 0.99 Å while for the active versus inactive βAR structures the RMSD is 0.85 Å. The RMSD values were computed over all non-hydrogen backbone and side-chain atoms in transmembrane helix residues that are in the union of (i) the LSD-binding pocket and the ERG-binding pocket for 5-HTR and (ii) the ICI 118,551-binding pocket and the BI-binding pocket for βAR (binding pocket residues are defined as those within 4.5 Å of the ligand in the crystal structure). We also observe conformational changes in the side chains of several important orthosteric pocket residues when comparing the structures of the LSD- and ERG-bound 5-HTR: T114, E363, and M218all change their rotamer states between the two structures ( Figure 2 A). These changes in rotamer states likely reflect distinct ligand-receptor interactions and an unexpected plasticity of the receptor for these structurally related compounds. For instance, in the 5-HTR/ERG complex, the phenyl moiety of ERG appears to “push” down on M218, wedging the M218side chain between the peptide and ergoline moiety of ERG ( Figure 2 A) and thus contributing to the deeper seating of the ergoline moiety of ERG in the pocket versus that of LSD. In the 5-HTR/LSD complex, by contrast, the diethylamide ergoline substituent does not interact with M218. As a result the M218side chain flips up, allowing more space for LSD to adopt a shallower binding mode. We also examined these differential binding modes by molecular dynamics (MD) simulations, which provided additional support for the hypothesis that the binding of LSD preserves the unliganded conformation of M218, whereas binding of ERG distorts it. In MD simulations initiated from structures of either the ERG-bound or LSD-bound 5-HTR but with the ligand removed, the M218side chain consistently adopted an upward conformation matching that of the LSD-bound structure ( Figure S3 A). We performed over 100 μs of simulation ( Table S1 ).

2B R/LSD and 5-HT 2B R/ERG complexes with CASTp ( Dundas et al., 2006 Dundas J.

Ouyang Z.

Tseng J.

Binkowski A.

Turpaz Y.

Liang J. CASTp: Computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. 3—a 28.6% decrease. Together, these data illustrate how distinct but similar compounds—in this case LSD and ERG—differentially and unexpectedly shape the ligand binding surface of a GPCR (i.e., 5-HT 2B R; 3.32 ( The smaller amide substituent of LSD also accounts for an overall contraction of the extended binding site relative to the ERG-bound structure ( Figure 2 B). Specifically, we observe an inward movement of helices II (1.6 Å), VII (2.1 Å), and parts of EL2 (1.0 Å) and EL3 (1.8 Å) toward the seven transmembrane core, and a relocation of helix VI (1.0 Å) away from helix VII toward helix V and the membrane, which is likely a result of the inward movement of helix VII ( Figure 2 B). Indeed, when we calculated the size of the binding pockets in the 5-HTR/LSD and 5-HTR/ERG complexes with CASTp (), we saw an overall reduction of the binding pocket volume from 2898.7 to 2068.4 Å—a 28.6% decrease. Together, these data illustrate how distinct but similar compounds—in this case LSD and ERG—differentially and unexpectedly shape the ligand binding surface of a GPCR (i.e., 5-HTR; Figures 2 C, 2D, S3 C, and S3D). We also observed that the amide substituents of LSD and ERG are differentially arranged with respect to the ionic bond with D135 Figure 2 A, inset).