Molecular determinants of allosteric modulation at the M1 muscarinic acetylcholine receptor

Abstract

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M1 muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M1 mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85(2.64) in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397(7.32) and Trp-400(7.35) in TMVII as residues that contribute to the BQCA binding pocket at the M1 mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described "common" allosteric site in the extracellular vestibule of the M1 mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102(3.29) and Asp-105(3.32)) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.

Keywords: Allosteric Regulation; Drug Discovery; G Protein-coupled Receptors (GPCR); Molecular Dynamics; Muscarinic Acetylcholine Receptor; Site-directed Mutagenesis.

FIGURE 1.

Mutations and ligands investigated in this study. A snake diagram of the human M₁ mAChR highlighting mutated residues and chemical structure of the allosteric modulator BQCA.

FIGURE 2.

Orthosteric agonist affinity estimates are differentially modified by M₁ mAChR mutations. Bars represent the difference in pK_A of orthosteric antagonist [³H]NMS or [³H]QNB (top panel) derived from whole cell saturation binding experiments (Table 1) or the difference in pK_I of the orthosteric agonist CCh (bottom panel) derived from whole cell competition binding experiments (Table 2), relative to the WT receptor value for each ligand at each mutant residue. Data represent the mean ± S.E. of three experiments performed in duplicate. *, significantly different from WT, p < 0.05, one-way analysis of variance, Dunnett's post hoc test.

FIGURE 3.

Identification of residues that differentially govern BQCA affinity and binding cooperativity with CCh at the M₁ mAChR. The curves represent competition between [³H]NMS (A–C, E, and F) or [³H]QNB (D) and increasing concentrations of CCh in the absence or presence of varying concentrations of BQCA. All assays were performed using 0.3 nm [³H]NMS or [³H]QNB in whole cells expressing the WT or mutant c-Myc-tagged M₁ mAChRs as described under “Experimental Procedures.” Data points represent the mean ± S.E. of three independent experiments performed in duplicate. Curves drawn through the points in A–C and F represent the best fit of an allosteric ternary complex model (Equation 1). Parameters obtained from these experiments are listed in Table 2.

FIGURE 4.

Effects of M₁ mAChR mutations on BQCA affinity and binding cooperativity estimates. Bars represent the difference in pK_B (top panel) or binding cooperativity value (log α, bottom panel) of BQCA relative to WT as derived from binding interaction experiments with CCh (Table 2). Data represent the mean ± S.E. of three experiments performed in duplicate. ND, no modulation by BQCA. *, significantly different from WT, p < 0.05, one-way analysis of variance, Dunnett's post hoc test.

FIGURE 5.

Positive correlation between the changes in orthosteric and allosteric ligand affinities at the M₁ mAChR mutants. Each point represents the affinity values of BQCA (pK_B) and CCh (pK_I) as determined from whole cell competition binding studies as listed in Table 2.

FIGURE 6.

CCh signaling efficacy (log τ_A) estimates are differentially affected by M₁ mAChR mutations. Bars represent the difference in log τ_A of CCh at each mutant relative to the WT receptor value, as derived from application of the operational model of allosterism to the IP₁ interaction data at each mutant (Equation 2). Data represent the mean ± S.E. of three experiments performed in duplicate. NR indicates that CCh activity was absent. ND indicates that Equation 2 could not be used due to loss of allosteric modulation by BQCA. *, significantly different to WT receptor value, p < 0.05, one-way analysis of variance, Dunnett's post hoc test.

FIGURE 7.

Identification of residues that differentially govern BQCA efficacy and functional cooperativity with CCh at the M₁ mAChR. Interaction between BQCA and CCh in IP₁ accumulation assay in CHO FlpIn cells stably expressing the WT or mutant M₁ mAChRs. Data points represent the mean ± S.E. of three independent experiments performed in duplicate. Curves drawn through the points in A, C, D, and F represent the best fit of an operational allosteric model (Equation 2 and Table 4) with the affinity of each ligand at each mutant fixed to the value determined from separate binding studies (Table 2).

FIGURE 8.

BQCA functional cooperativities are differentially modified by M₁ mAChR mutations. Bars represent the difference in the functional cooperativity value (log αβ, Equation 2) relative to the WT value, as derived from application of the operational model of allosterism to the CCh and BQCA IP₁ interaction data at each mutant (Equation 2) (Table 4). Data represent the mean ± S.E. from three experiments performed in duplicate. ND, no modulation by BQCA. * significantly different from WT value, p < 0.05, one-way analysis of variance, Dunnett's post test.

FIGURE 9.

Structural model of the M₁ mAChR in complex with BQCA and CCh. A, overall view of the complex obtained using MD simulations. The ligands are shown in orange (BQCA) and yellow (CCh) spheres. B, orthosteric binding site for CCh; C, predicted allosteric binding site of BQCA. Important residues are shown by orange sticks.

FIGURE 10.

Proposed rearrangement of ECLs and TMs upon BQCA binding to M₁ mAChR. Extracellular view of the BQC-binding site at the starting position for MD simulations (gray) or at the final position of the receptor after 20 ns of MD (orange). Important residues involved in BQCA binding or cooperativity are shown as sticks. Global movements of TMs and ECLs are shown with green arrows, and residue shifts are indicated by blue arrows. Inset shows the movement of the side chain of Trp-400^7.35.