(A) Chemical structures of 5-HT, LSD and KET. Amines interacting with D3.32, S3.36 or S5.46 [6], [7], [116], [117], [118], [119] are labeled. (B,C,D) Docking poses in the initial structures (left panels) and during the simulations (right panels) for 5-HT (B), LSD (C) and KET (D), respectively. For clarity, only TM 3, 5 and 6 are shown in grey ribbons. Sidechains of residues D3.32, S3.36, S5.43, S5.46, F5.47, F6.44, W6.48, F6.51, F6.52 and N6.55 are depicted as sticks, and 5-HT (carbons colored in orange), LSD (cyan) and KET (green) are rendered in spheres. Note that, due to its large-size, and because its quinazoline ring penetrates deep into the binding pocket close to W6.48, KET is in direct contact with all the residues in the aromatic cluster, including F5.47. (E) Time-evolution of backbone TM RMSDs of 5-HT_2AR (upper panel) and of the distances between the carboxyl/hydroxyl oxygens in D3.32, S3.36 and S5.46 on 5-HT_2AR and their interacting amine nitrogens on ligands (see panel A) during the simulations (lower panels). Traces are shown in orange for 5-HT, in cyan for LSD, and in green for KET. Data were collected every 100 ps. Running averages were calculated every 10 data points and are shown in bold shades. N_α atom of 5-HT maintains a salt-bridge with D3.32 and forms an H-bond with S3.36 (Figure S2 in Text S1); N₁ atom of 5-HT forms an H-bond with S5.46 either directly or through a water-bridge.

(A–E) Left and right panels show the evolution of active state components in the 5-HT_2AR complexed with LSD and KET, respectively (for details see Figure 3). (F) Cartoon representation of TM3 and TM6 in the structures averaged over the last 100 ns of the LSD (cyan) and KET (green) trajectories, showing positions of R3.50 and E6.30 residues (in sticks).

(A) Projections along the first and second eigenvectors obtained from the Comb-ED analysis on the concatenated 5-HT-LSD (upper panel), 5-HT-KET (middle panel), and LSD-KET (lower panel) trajectories. The centers of the conformational space sampled by ligands are in black dots and are connected by black dotted lines. (B) Extreme projections along the first eigenvector of the combined 5-HT-LSD (top panel), 5-HT-KET (middle panel) and LSD-KET (bottom panel) trajectories. The receptor is rendered and colored as in Figure 5B. (C) Comparison of the 5-HT_2AR structures in complex with 5-HT, LSD or KET averaged over the final 100 ns aligned with seven most conserved residues in each TM [18]. The receptor structures in complex with different ligands are shown in cartoon and are colored as in panel A.

The structures of the various ligand-bound receptor structures averaged over the last 100 ns of the simulations are shown in cartoon, with only the helices depicted (in different colors) with corresponding TM numbers. The colored fields represent distances (in Å) between lipid backbone C₂ atoms from the opposing leaflets. For this analysis, for each simulated system the membrane plane was divided into square 2 Å×2 Å bins, and the average C₂-C₂ distances in each bin were collected by scanning the last 100 ns of trajectory.

(A) Known structural elements of GPCR activation (SM/FM) in the homology model of the 5-HT_2AR. (B) The time-ordered sequence of events identified from the MD simulations of the agonist-bound 5-HT_2AR.

(A) Evolution of the bend angle in TM6 around P6.50, highlighting the intervals during which the helix straightens (event 1) and bends (event 3) upon activation. (B) Evolution of the C_α distances between D3.32 and F6.52 (red), and between D3.32 and N6.55 (black), illustrating the interval during which the EC end of TM6 moves towards TM3 (event 2). (C) Evolution of the tilt angle of the toggle switch W6.48 aromatic ring with respect to the membrane normal, showing the time point of W6.48 flipping (event 4). W6.48 becomes parallel to the membrane for ∼1 ns at 143 ns (see panel J) with χ₁ angle changing from g- to trans. (D) Evolution of the C_α distance between D3.32 and V6.40 illustrating the interval when the IC end of TM6 moves away from TM3 (event 5). (E) Dynamics of the ionic lock presented as the evolution of the C_α distance between R3.50 and E6.30. Initially broken ionic lock forms during the first 50 ns, before opening again upon activation at ∼170 ns (event 6). (F) Evolution of the C_α distance between Y7.53 and Y7.60. (G) Snapshots from the membrane plane and the EC end, highlighting positions of D3.32 and N6.55 and the distance between them, and showing the initial straightening and motion of TM6 towards TM3 (event 1). Gray cartoon represents the starting structure, and the orange cartoon is the structure averaged over the 83–112 ns interval. (H) Cartoon representation of TM3 and TM6 highlighting the kink in the TM6 that occurs in the 135–225 ns time interval (event 3). Orange and Magenta cartoons represent structures averaged over 83–112 ns and 290–350 ns, respectively. (I) Snapshots of TM3 and TM6 depicting positions of R3.50 and E6.30 residues and the distance between them and illustrating the movement of TM6 away from TM3 (event 5). Color code is the same as in panel G. (J) Detailed dynamics in the toggle switch W6.48. Evolution of the χ₁ and χ₂ angles is shown during the 140–146 ns time-interval when the toggle switch flips. Also shown are the snapshots at 0 ns and 143 ns time-points of the 5-HT and W6.48 (in spheres and colored by atom type, 5-HT in orange, and W6.48 in grey and cyan at 0 ns and 143 ns, respectively.).

(A) Evolution of the TM backbone RMSD of KET-substituted receptor compared to KET-bound receptor, averaged along 250–350 ns (top), the minimum distance between the carboxylate oxygens of D3.49 and the guanidine nitrogens of R3.50 (middle), as well as the C_α distance between R3.50 and E6.30 (bottom). (B) Extreme projections along the first eigenvector from Comb-ED analysis of the combined 5-HT-bound and KET-substituted receptors (left panel), as well as KET-bound and KET-substituted (right panel) trajectories. The receptor is shown in tubes, and colors depict magnitudes of conformational changes from small to large (from blue to green, and to red).

(A) Evolution of the minimum distances between the Chol at the EC end of TM6 and selected TM6 residues in the 5-HT simulation (top panel). Time traces of the minimum distances between the Chol at the IC ends of TM6–7 and selected residues on TM6 and 7 (middle and bottom panels). The Chol initially in contact with the L7.44, V7.48, V7.52, and L7.55 residues on TM7 moves towards TM6 and engages in interactions with the residues K6.35, I6.39, F6.42, and V6.46 on TM6. (B) Snapshots at 10 and 30 ns showing the Chol from the top panel of (A) interacting with EC TM6. (C) Snapshots at 50, 167.6 and 250 ns showing the Chol from the bottom panels of (A) interacting with either IC TM6 or IC TM7. (D) Matrix of Pearson's score tests performed on the dynamics quantities presented in the top panel of (A) and on the bend (“B”) and face-shift (“FS”) angles around P6.50 and P7.50 (top panel). Matrix of Pearson's score tests performed on the dynamic quantities presented in middle and bottom panels of (A) and on the bend (“B”), and face-shift (“FS”) angles of P6.50 and P7.50 (bottom panel).