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. 2023 May 30;12:e83477. doi: 10.7554/eLife.83477

Figure 4. Binding and dynamics of LY298 and VU154.

(A, B) Cryo-electron microscopy (cryo-EM) density of the (A) VU154- and (B) LY298-binding sites. (C) The root mean square deviations (RMSDs) between receptor models of the respective cryo-EM structures that were refined into the first and last frames of the EM maps from each principal component (PC1-PC3) of the 3D variability analysis. Values shown are mean ± SEM. (D, E) Top representative binding conformations of (D) VU154 and (E) LY298 obtained from structural clustering with frame populations ≥1% and time courses of the RMSDs of each positive allosteric modulator (PAM) relative to the cryo-EM structures. (F, G) Binding interactions of VU154 and LY298 with views from the (F) membrane and (G) extracellular surface. (H) Position and χ2 angle of W4357.35 in the tiotropium-, ACh-, Ipx-, VU154-Ipx-, and LY298-Ipx bound structures. (I–K) Time courses of the W4357.35 χ2 angle obtained from Gaussian accelerated molecular dynamics (GaMD) simulations on the (I) Ipx-, (J) VU154-Ipx-, and (K) LY298-Ipx-bound cryo-EM structures. See Table 3.

Figure 4.

Figure 4—figure supplement 1. Gaussian accelerated molecular dynamics (GaMD) simulations of LY293 and VU154 binding.

Figure 4—figure supplement 1.

(A–K) Time courses from three 500 ns GaMD simulations using the (A–D) VU154-Ipx- and (E–H) LY298-Ipx-bound cryo-electron microscopy (cryo-EM) structures. Distances between the interactions of VU154 and LY298 with residues (A, E) Y897.39, (B, F) F18645.51, (C, G) Y4397.39, and (D, H) Q18445.49. (I, J) Distance between (I) Y922.64 and (J) T4337.33 to VU154 from GaMD simulations of the VU154-Ipx-M4R-Gi1 structure. (K) Distance between N4236.58 and the fluorine atom of LY298 from GaMD simulations of the LY298-Ipx-M4R-Gi1 structure. See Table 3.
Figure 4—figure supplement 2. Key residues for the binding of LY298 and VU154 at the human M4 muscarinic acetylcholine receptor (mAChR).

Figure 4—figure supplement 2.

(A, B) Competition binding with a fixed concentration of [3H]-NMS and increasing concentrations of acetylcholine (ACh) (black circles), (A) LY298 or (B) VU154 (blue circles), and LY298 or VU154 in the presence of an IC20 concentration of ACh (red squares). Curves drawn through the points represent a global fit of an extended ternary complex model. Data points represent the mean ± SEM of three or more independent experiments performed in duplicate. Similar data were observed for competition binding with iperoxo (Ipx) instead of ACh. See Table 4.
Figure 4—figure supplement 2—source data 1. Related to Figure 4—figure supplement 2.
Figure 4—figure supplement 3. Gaussian accelerated molecular dynamics (GaMD) simulations of M4R complexes with acetylcholine (ACh).

Figure 4—figure supplement 3.

(A–L) Time courses from GaMD simulations, each performed with three separate replicates. Individual replicate simulations are illustrated with different colors. The heading of each plot refers to the specific model used in the simulations. See Table 3. (A–C) Root mean square deviations (RMSDs) of ACh from simulations of the (A) cryo-electron microscopy (cryo-EM) structure or (B, C) positive allosteric modulator (PAM) docked models. (D, E) RMSDs of VU154 and LY298 from the ACh-bound M4 mAChR simulations. (F) Bar graph of the root mean fluctuations of the agonists iperoxo (Ipx) or ACh across the GaMD simulations of the M4-Gi1 complexes with or without the PAMs. Values shown are mean ± SEM, n = 3. (G–L) Time course of the ACh-bound M4-Gi1 simulations illustrating variances in the (G–I) W4357.35 χ2 angle and (J–L) the W4136.48 χ2 angle.