Cryo-EM reveals a new allosteric binding site at the M5 mAChR

Abstract

The M₅ muscarinic acetylcholine receptor (M₅ mAChR) represents a promising therapeutic target for neurological disorders. However, the high conservation of its orthosteric binding site has posed significant challenges for drug development. While selective positive allosteric modulators (PAMs) offer a potential solution, a structural understanding of the M₅ mAChR and its allosteric binding sites has remained limited. Here, we present a 2.8 Å cryo-electron microscopy structure of the M₅ mAChR complexed with heterotrimeric G_q protein and the agonist iperoxo, completing the active-state structural characterization of the mAChR family. To identify the binding site of M₅-selective PAMs, we implemented an integrated approach combining mutagenesis, pharmacological assays, structural biology, and molecular dynamics simulations. Our mutagenesis studies revealed that selective M₅ PAMs bind outside previously characterized M₅ mAChR allosteric sites. Subsequently, we obtained a 2.1 Å structure of M₅ mAChR co-bound with acetylcholine and the selective PAM VU6007678, revealing a novel allosteric pocket at the extrahelical interface between transmembrane domains 3 and 4 that was confirmed through mutagenesis and simulations. These findings demonstrate the diverse mechanisms of allosteric regulation in mAChRs and highlight the value of integrating pharmacological and structural approaches to identify novel allosteric binding sites.

Figure 1.. Structural and functional analysis of ligand-receptor interactions at the M₅ mAChR.

(A) Interaction of ML380 with ACh in an IP1accumulation assay at WT M₅ mAChR (left) or M₅ EH4 pocket mutant mAChR (right) expressing CHO cells. Data points represent mean ± SEM of three to seven individual experiments performed in duplicate. WT M₅ mAChR n = 7, M₅ EH4 pocket mutant n = 3. An operational model of allosterism was fit to the data. (B) Effects of the M₅ mAChR mutations on the pK_B of ML380. Data represent the mean ± SEM of three to seven independent experiments performed in duplicate. WT M₅ mAChR n = 7, all other mutants n = 3. *, significantly different from WT, p < 0.05, one-way ANOVA, Dunnett’s post hoc test. Parameters obtained are listed in Supplementary Table 1. (C) M₅ mAChR mutated residues are shown on the structure of the M₅ mAChR. (D) Residues of the EH4 pocket are shown as purple sticks. (E) Residues of the ‘common’ ECV are shown as red sticks. (F) Consensus cryo-EM map of the M₅ mAChR in complex with Gα_mGsQi/Gβ₁γ₂ bound to iperoxo resolved to 2.8 Å (FSC 0.143). The receptor is shown in dark blue, the heterotrimeric G_q protein is shown in red, green, and yellow for the α, β, γ subunits, respectively. (G) Cryo-EM density (local refined receptor map, contour level 0.36) for iperoxo in the orthosteric binding site. (H) Interactions of iperoxo with the orthosteric binding site. Charge-charge interactions are shown as pink dotted lines, hydrogen bonds are shown as black dotted lines while purple dotted lines represent a cation-π interaction with Y111^3.33, W455^6.48, Y458^6.51, and Y481^7.39.

Figure 2:. Structural comparison of iperoxo bound, active state M₁-M₅ mAChR structures determined by cryo-EM.

(A) Overall view of the M₁ to M₅ mAChRs complexed to Gα and bound to iperoxo. M₁-iperoxo-Gα11 is coloured purple, M₂-iperoxo-Gα_oA is coloured orange, M₃-iperoxo-Gα_mGsQi is coloured light blue, M₄-iperoxo-dnGα_i1 is coloured red, M₅-iperoxo- Gα_mGsQi is coloured dark blue. (B) Extracellular view comparing ECLs and TM regions across the M₁ to M₅ mAChRs. (C) Intracellular view (G protein removed) comparing ICLs and TM regions across the M₁ to M₅ mAChRs. (D) Overlay of iperoxo and orthosteric binding site residues at the M₁ to M₅ mAChRs. (E) Intracellular view comparing the αN movement of Gα at M₁ to M₅ mAChRs. Changes in orientation is indicated by an arrow. (F) Intracellular view comparing the α5 insertion of Gα into the M₁ to M₅ mAChRs. Changes in insertion is indicated by an arrow. (G) Intracellular view comparing the α5 rotation of Gα relative to M₁ to M₅ mAChRs. Changes in rotation is indicated by an arrow.

Figure 3:. Potential cryo-EM density for ML380.

(A-C) Local refined receptor cryo-EM map of the M₅ mAChR (contour level 0.3). No density for ML380 was observed in the (A) ‘common’ ECV (B) or in the EH4 pocket. (C) potential density for ML380 was observed parallel to TM1 and TM7 (coloured green) and at the bottom of the TM2,3,4 interface (coloured orange). (D) [³H]-NMS equilibrium radioligand binding studies between [³H]-NMS, ACh and ML380 at the WT M₅ mAChR and M₅-M₂ TM chimeric swaps. The insets are cartoons of the WT M₅ mAChR and M₅-M₂ TM chimeric swaps with blue representing M₅ mAChR domains and red representing M₂ mAChR domains. Data points represent mean ± SEM of three individual experiments performed in duplicate. Data was fit to an allosteric ternary complex model. Parameters obtained are listed in Supplementary Table 3. (E-F) Root mean square deviations (RMSDs) of (E) iperoxo in the orthosteric binding pocket and (F) ML380 at the potential allosteric binding site at the bottom of the TM2,3,4 interface calculated from Gaussian accelerated molecular dynamics (GaMD) simulations of the cryo-EM structure, each performed with three separate replicates indicated with different coloured traces.

Figure 4:. High resolution structure of ACh and VU6007678 bound M₅ mAChR in complex with heterotrimeric G protein.

(A) Consensus cryo-EM map of the M₅ mAChR in complex with Gα_mGsQi/Gβ₁γ₂ bound to ACh and VU6007678 resolved to 2.1 Å (FSC 0.143). The receptor is shown in light blue, the heterotrimeric G_q protein is shown in red, green, and yellow for the α, β, γ subunits, respectively, scFv16 is shown in light grey and Nb35 is shown in dark grey. (B) Model of the M₅ mAChR showing ACh (green) in the orthosteric binding site and VU6007678 (orange) in an allosteric binding site at the bottom at TM3 and 4 and on top of ICL2. (C) Cryo-EM density (local refined receptor map, contour level 0.43) for iperoxo in the orthosteric binding site. (D) Cryo-EM density (local refined receptor map, contour level 0.43) for VU6007678 in an allosteric binding site. (E) Comparison of the orthosteric binding site interactions at ACh and Ipx-bound M₅ mAChR. Shown in light blue sticks is ACh-bound M₅ mAChR residues, dark blue sticks is Ipx-bound M₅ mAChR residues. ACh is shown in light green sticks and Ipx is shown in dark green sticks. (F) Interactions of ACh with the orthosteric binding site of the M₅ mAChR. Charge-charge interactions are shown as pink dotted lines, hydrogen bonds are shown as black dotted lines while purple dotted lines represent a cation-π interaction with Y111^3.33, W455^6.48, Y458^6.51, and Y481^7.39. Red spheres indicate water molecules. (G) 2D interaction plot of ACh with the orthosteric binding site of the M₅ mAChR. Charge-charge interactions are shown as pink dashed lines, hydrogen bonds are shown as black dashed lines, cation-π interactions are shown as purple dashed lines and hydrophobic interactions are shown as a solid orange line. (H) RMSDs of ACh relative to the starting conformation in the orthosteric pocket calculated from GaMD simulations of the cryo-EM structure performed with three separate replicates indicated through different coloured traces. (I) Interactions of VU6007678 with its allosteric binding site at the M₅ mAChR. Hydrogen bonds are shown as black dotted lines, while purple dotted lines represent cation-π interactions, and green dotted lines represent π-π interactions. (J) Comparison of the VU6007678 allosteric binding site to the inactive M₅ mAChR when bound to the orthosteric antagonist tiotropium (PDB:6OL9). VU6007678 is shown in orange, the M₅ mAChR bound to ACh and VU6007678 in light blue and the inactive tiotropium bound M₅ mAChR in salmon. (K) 2D interaction plot of VU6007678 with the its allosteric binding site. Hydrogen bonds are shown as black dashed lines, while purple dashed lines represent cation-π interactions, and green dashed lines represent π-π interactions. (L) RMSDs of VU6007678 relative to the starting conformation at the allosteric binding site calculated from GaMD simulations of the cryo-EM structure performed with three separate replicates indicated through different coloured traces.

Figure 5:. Structural and functional analysis of the VU6007678 allosteric binding site.

(A) Comparison of VU6007678 allosteric binding site residues (stick representation) across the M₁-M₅ mAChRs. M₁ mAChR (PDB: 6OIJ) is shown in purple, M₂ mAChR (PDB: 6OIK) is shown in orange, M₃ mAChR (PDB: 8E9Z) is shown in light blue, M₄ mAChR (PDB: 7TRK) is shown in red and the M₅ mAChR is shown in dark blue. (B-C) Interaction of ML380 with ACh in Trupath G protein activation assay in (B) WT M₅ mAChR or (C) M₅/M₂ swap expressing CHO cells. Data points represent mean ± SEM of three to eight individual experiments performed in duplicate. WT M₅ mAChR n = 8, M₅/M₂ swap mutant n = 3. An operational model of allosterism was fit to the data. (D-F) Key pharmacological parameters for the interaction of ACh and VU6007678 in Gα_q activation at WT M₅ mAChR and mutants. Data points represent mean ± SEM of three to eight individual experiments performed in duplicate. WT M₅ mAChR n = 8, M₅/M₂ swap, F130^3.52M, T133^3.55A, R134^3.56A, R134^3.56K mutants n = 3, Y68^2.42F, V123^3.45I, K141^ICL2A, R146^4.41M mutants n = 4. An operational model of allosterism was fit to the data. Parameters obtained are listed in Supplementary Table 4. (G) Affinity of VU6007678 (pK_B) and (H) logarithm of affinity cooperativity (logα) between the orthosteric agonist (ACh) and allosteric modulator (VU6007876) at the WT M₅ mAChR and selected mutants obtained in [³H]-NMS equilibrium radioligand binding studies between ACh, VU6007678 and [³H]-NMS. Data points represent mean ± SEM of three to five individual experiments performed in duplicate. WT M₅ mAChR n = 3, F130^3.52M, R146^4.41M n = 4, M₅/M₂ swap n = 5. Data was fit to an allosteric ternary complex model. Parameters obtained listed in Supplementary Table 4. (I-K) Time courses of distances from VU6007678 to (I) F130^3.52, (J) R146^4.41, (K) M150^4.45 in Å calculated from GaMD simulations performed with three separate replicates as indicated through different coloured traces.

Figure 6:. Allosteric sites at the mAChRs.

(A) Model of the ACh-VU6007678 bound M₅ mAChR showing the novel allosteric binding site discovered for VU6007678 (orange spheres) and the ‘common’ ECV allosteric site occupied by LY2119620 (salmon spheres). ACh is shown as green spheres in the orthosteric binding site. (B) Cryo-EM density (contour level 0.47) for lipid molecules observed at the bottom of TM2,3,4 in the orthosteric binding site.