Journal: Nature Communications

Article Title: Cryo-EM reveals an extrahelical allosteric binding site at the M 5 mAChR

doi: 10.1038/s41467-025-62212-z

Figure Lengend Snippet: a–c Local refined M 5 mAChR cryo-EM map (contour level 0.3). No ML380 density was observed in a ECV or b EH4 pocket. c Potential ML380 density observed parallel to TM1/TM7 (green) and the bottom of the TM2,3,4 interface (orange). d [ H]-NMS equilibrium binding studies between [ H]-NMS, ACh and ML380 at WT M 5 mAChR and M 5 -M 2 TM chimeras. Insets show receptor cartoons (blue: M 5 domains; red: M 2 domains). Data points are mean ± SEM values of three duplicate experiments. An allosteric ternary complex model was fit to the data. Parameters obtained are listed in Supplementary Table . RMSDs (Å) of e iperoxo in orthosteric pocket and f ML380 at the potential allosteric site at the bottom of the TM2,3,4 interface from GaMD simulations of the cryo-EM structure. Three simulation replicates are shown in different colours. Source data are provided as a Source Data file.

Article Snippet: For further analysis, we performed all-atom Gaussian accelerated Molecular Dynamics (GaMD) simulations on the modelled structure of the iperoxo-M 5 mAChR-mGα q complex with ML380 bound at the TM2,3,4 interface (Fig. ).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature Communications

Article Title: Cryo-EM reveals an extrahelical allosteric binding site at the M 5 mAChR

doi: 10.1038/s41467-025-62212-z

Figure Lengend Snippet: a Consensus cryo-EM map of M 5 mAChR-mGα q /Gβ 1 γ 2 complex with ACh and VU6007678 at 2.1 Å resolution (FSC 0.143). Model colouring: light blue (receptor); red (mGα q ), green (Gβ 1 ), yellow (Gγ 1 ), light grey (scFv16), dark grey (Nb35). b M 5 mAChR model showing ACh (green) at the orthosteric site and VU6007678 (orange) at the allosteric site between TM3/TM4 and above ICL2. Cryo-EM density (local refined receptor map, contour 0.43) for c iperoxo at the orthosteric site and d VU6007678 at the allosteric site. e Comparison of orthosteric site residues. Stick colouring: light blue (ACh-bound residues), dark blue (Ipx-bound residues), light green (ACh), dark green (Ipx). f Interactions of ACh with the orthosteric binding site. Interaction colouring: pink dotted lines (charge-charge), black (hydrogen bonds), purple (cation-π); red spheres (water molecules). g 2D interaction plot of ACh with the orthosteric binding site; grey lines (hydrophobic interactions). h RMSDs (Å) of ACh relative to the starting conformation during GaMD simulations (three replicates, different colours). i VU6007678 allosteric site interactions: black (hydrogen bonds), purple (cation-π), green (π-π). j VU6007678 site comparison between the active complex (light blue) and inactive tiotropium-bound M 5 (salmon, PDB:6OL9); VU6007678 (orange). k 2D VU6007678 interaction plot. Same colouring as g with green dashed lines representing π-π interactions. l RMSDs (Å) of VU6007678 relative to the starting conformation during GaMD simulations (three replicates, different colours). Source data are provided as a Source Data file.

Article Snippet: For further analysis, we performed all-atom Gaussian accelerated Molecular Dynamics (GaMD) simulations on the modelled structure of the iperoxo-M 5 mAChR-mGα q complex with ML380 bound at the TM2,3,4 interface (Fig. ).

Techniques: Cryo-EM Sample Prep, Comparison, Binding Assay

Journal: Nature Communications

Article Title: Cryo-EM reveals an extrahelical allosteric binding site at the M 5 mAChR

doi: 10.1038/s41467-025-62212-z

Figure Lengend Snippet: a Comparison of VU6007678 allosteric binding site residues (stick representation) across the M 1 -M 5 mAChRs. Colouring: purple (M 1 ; PDB: 6OIJ), orange (M 2 ; PDB: 6OIK), light blue (M 3 ; PDB: 8E9Z), red (M ; PDB: 7TRK), and dark blue (M 5 ; PDB: 9EJZ). VU6007678-ACh interaction in Trupath G q activation assay at b WT M 5 mAChR or c M 5 /M 2 swap in CHO cells. Data points are mean ± SEM of three to eight individual experiments performed in duplicate. WT M 5 mAChR n = 8, M 5 /M 2 swap mutant n = 3. An operational model of allosterism was fitted to the grouped data to derive the key pharmacological parameters ( d – f ). Pharmacology parameter values were shared across experiments to yield a single best estimate of the mean of each parameter and its associated standard error, as derived from the nonlinear regression algorithm. This global pooled analysis approach ensured model convergence in all instances. *, significantly different from WT, p < 0.05, one-way ANOVA, Dunnett’s post hoc test. WT M 5 mAChR n = 8; M 5 /M 2 swap, F130 3.52 M, T133 3.55 A, R134 3.56 A, R134 3.56 K mutants n = 3; Y68 2.42 F, V123 3.45 I, K141 ICL2 A, R146 4.41 M mutants n = 4. g VU6007678 affinity (pK B ) and h log affinity cooperativity (logαβ) between ACh and VU6007876 at WT M 5 mAChR and mutants from [ 3 H]-NMS equilibrium binding studies. Data points represent mean ± SEM values of pharmacological parameters determined by fitting an allosteric ternary complex model to the grouped data, as described in ( d – f ). All pharmacology parameters ( d – h ) are listed in Supplementary Table . WT M 5 mAChR n = 3, F130 3.52 M, R146 4.41 M n = 4, M 5 /M 2 swap n = 5. Time courses of distances (Å) from VU6007678 to i F130 3.52 , j R146 4.41 , k M150 4.45 calculated from GaMD simulations performed with three separate replicates as indicated through different coloured traces. Source data are provided as a Source Data file.

Article Snippet: For further analysis, we performed all-atom Gaussian accelerated Molecular Dynamics (GaMD) simulations on the modelled structure of the iperoxo-M 5 mAChR-mGα q complex with ML380 bound at the TM2,3,4 interface (Fig. ).

Techniques: Comparison, Binding Assay, Activation Assay, Mutagenesis, Derivative Assay