Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

Abstract

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

Figure 1

The M₂ receptor (blue ribbon) with bound QNB (orange spheres). a, M₂ receptor in profile. b, Cytoplasmic surface showing conserved DRY residues in TM3. c, Extracellular view into QNB binding pocket. d, Extracellular view with solvent-accessible surface rendering shows a funnel-shaped vestibule and a nearly buried QNB binding pocket. e, Aqueous channel (green) extending from the extracellular surface into the transmembrane core is interrupted by a layer of three hydrophobic residues (blue spheres). Well-ordered water molecules are shown as red dots.

Figure 2

Binding interactions between the M₂ receptor and QNB. a, b, Two views of the QNB binding pocket. Amino acids within 4 Å of the ligand are shown as light blue sticks, with QNB in orange. Nitrogen and oxygen atoms are colored dark blue and red, respectively. Polar interactions are indicated by dashed lines. A 2F_o–F_c map is shown in wire at 1.5 σ contour. c, A schematic representation of QNB binding interactions is shown. Mutations of amino acids in red boxes have been shown to reduce both antagonist and agonist binding by more than 10 fold. Mutations of the amino acid in the purple boxes reduce antagonist binding affinity by more that 10 fold. Mutations of amino acids in blue boxes reduce agonist binding by more than 10 fold. Blue dotted lines indicate potential hydrophobic interactions and red lines indicate potential polar interactions.

Figure 3

Convergent evolution of acetylcholine binding sites. a, Acetylcholine is modeled into the crystal structure of the M₂ receptor. b, Acetylcholine binding pocket in the crystal structure of the acetylcholine binding protein from the snail Aplysia californica (PDB ID: 2XZ5). c, Acetylcholine binding pocket in the acetylcholine binding protein ChoX from the gram negative bacterium Sinorhizobium meliloti (PDB ID: 2RIN). d, Binding site for thio-acetylcholine in the enzyme acetylcholine esterase from the electric ray Torpedo californica (PDB ID: 2C4H).

Figure 4

Allosteric binding in the M₂ receptor. a, Differences between the M₂ and M₄ receptors are shown as green residues mapped onto the inner surface of the M₂ receptor (blue), with QNB in orange spheres. The sequence conservation within the orthosteric site is apparent, while residues outside show more variability. b–d, Mutations that alter allosteric binding are shown with yellow carbons, and amino acids involved in QNB binding are shown with blue carbons as sticks or spheres. b, c, Different views of possible allosteric binding sites in the M₂ receptor. The surface view in c shows the positions of possible allosteric binding sites (yellow) lining the path to the QNB binding pocket. d, Trp422 (yellow spheres), implicated in binding of allosteric ligands, forms an edge-to-face aromatic interaction with Tyr403, part of the aromatic cage (blue spheres) of the orthosteric site.