Fig 1.
Important features and ligands in the available experimental structures of the 5-HT2AR.
(A) The serotonin-2A receptor with important side chains highlighted. (B) The intracellular side of 5-HT2AR. In red the conformation in its active form and in blue its inactive one. The two arrows represent the intracellular helix distances between TM2-6 and TM3-7, respectivally. Two important distances to discriminate between states (C) An overview of the ligands used for MD simulations.
Table 1.
Overview of the molecular dynamics (MD) simulations performed and the ligands used. Simulations are grouped as non-G-protein coupled (i.e., without the G protein construct; simulations 1–8) and G-protein-coupled (with the G protein construct; simulations 9–15). The PDB-template structures from which each simulation was initiated are also indicated and color-coded according to their inactive (blue) or active (red) receptor state.
Fig 2.
(A–B) Loadings for each DOF (as defined in S2 Table) for the first two principal components.The eigenvalues of the covariance matrix are 10.8 and 3.9, respectively, with explained variance ratios of 34% and 12% for PC1 and PC2. (C) Projection of simulations 1–15 and corresponding X-ray/cryo-EM structures onto the first two principal components.
Fig 3.
Time series showing the TM2–6 distance and the RMSD of the NPxxY motif relative to the inactive state.
Panels (A–C) present trajectories from simulations 2b, 5, and 7. Panels (D–F) show the corresponding trajectories for G protein–coupled systems from simulations 10, 12, and 14.
Fig 4.
Analysis of side chain conformations of microswitches.
χ1 and χ2 angles of (A) the W3366x48 switch and (B) the F3326x44.(C) Overlap between an “on” tryptophan switch (cyan) with the hallucinogen, an “off” one (orange) with antagonist zotepine and an “alternative” rotamer (purple) with (R)-69. (D) An active and inactive state of the PIF motif with F3326x44 a gauche and anti-like aconformation. (E) Blocked “Trp-switch” with antagonist zotepine.
Fig 5.
Analysis of important inter-side chain distances as identified by the PCA.
(A-B) 5-HT2A with (R)-69 and 25CN-NBOH, resp.Normalized histograms are shown for simulations 1-8 without the G protein (top row) and simulations 9-15 with the G protein construct (bottom row). (C) Distance between indole-nitrogen of W3366x48 and amide-oxygen of N3767x49. (D) Distance between hydroxyl-oxygens of Y2545x58 and Y3807x53.
Fig 6.
Analysis of important ligand-side chain interactions.
(A) Structure of 5-HT2A bound to (R)-69, highlighting the five additional DOFs included in the second PCA, along with the TM5 bulge and residue I1633x40.(B) TM5 bulge, quantified as the distance between residues S2425x46 and G3697x41. (C) Structures of 5-HT2A bound to the β-arrestin–biased ligands IHCH-7086 (top) and 25CN-NBOH (bottom).
Fig 7.
Boltzmann-inverted density plots for TM6–2 and TM3–7 axes, presented in free-energy units (kcal/mol).
Panel A shows simulations conducted without the G protein construct, while panel B displays simulations with the G protein construct present. Arrows indicate the direction of conformational transitions starting from the experimentally determined structures (denoted by stars).
Fig 8.
Contour plot depicting 50% most sampled TM6-2 and TM3-7 distances across simulations 1-15.
Colored stars represents the state their corresponding X-ray or Cryo-EM structures. It shows the distinct conformational regions (labeled by the 5-HT2A’s state) modulated by different ligands. A putative “axis of activation” is drawn in red along the TM6-2 and TM3-7 axes.