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

Haga K, Kruse AC, Asada H, Yurugi-Kobayashi T, Shiroishi M, Zhang C, Weis WI, Okada T, Kobilka BK, Haga T, Kobayashi T - Nature (2012)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Faculty of Science, Gakushuin University, Mejiro 1-5-1, Tokyo 171-8588, Japan.

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.

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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 2Fo–Fc 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.
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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 2Fo–Fc 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.

Mentions: The ligand QNB binds within a deeply buried pocket defined by side chains of TM3, 4, 5, 6 and 7 (Fig. 2a–c, Supplementary Fig. 5, Supplementary table 3). An aromatic cage encloses the amine and forms a lid over the ligand, separating the orthosteric site from the extracellular vestibule. Asp1033.32 and Asn4046.52 serve to orient the ligand in the largely hydrophobic binding cavity, with Asn4046.52 forming paired hydrogen bonds with the hydroxyl and carbonyl groups in QNB while Asp1033.32 engages in a charge-charge interaction with the amine moiety of the ligand (Fig. 2). The transmembrane segment amino acids that form the QNB binding pocket are identical in all five muscarinic receptor subtypes (Supplementary Table 1), consistent with results of QNB binding experiments on M1–M4 receptors, and with site-directed mutagenesis experiments on M119, M220, and M321 receptors. Only Phe181, which extends downward from ECL2 and interacts with one of the two phenyl rings on QNB (Fig. 2), differs from all other muscarinic receptor subtypes which have leucine in the homologous position. The importance of Asp3.32 for both agonist and antagonist binding has been demonstrated in mutagenesis and covalent-labeling experiments and modeling studies19–22. In contrast, mutation of Asn4046.52 to Ala on M123 and M324 receptors was shown to greatly affect binding of QNB but have little effect on binding of or activation by acetylcholine. It is possible that Asn4046.52 is hydrogen-bonded with ester group of QNB but not of acetylcholine.


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

Haga K, Kruse AC, Asada H, Yurugi-Kobayashi T, Shiroishi M, Zhang C, Weis WI, Okada T, Kobilka BK, Haga T, Kobayashi T - Nature (2012)

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 2Fo–Fc 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3345277&req=5

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 2Fo–Fc 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.
Mentions: The ligand QNB binds within a deeply buried pocket defined by side chains of TM3, 4, 5, 6 and 7 (Fig. 2a–c, Supplementary Fig. 5, Supplementary table 3). An aromatic cage encloses the amine and forms a lid over the ligand, separating the orthosteric site from the extracellular vestibule. Asp1033.32 and Asn4046.52 serve to orient the ligand in the largely hydrophobic binding cavity, with Asn4046.52 forming paired hydrogen bonds with the hydroxyl and carbonyl groups in QNB while Asp1033.32 engages in a charge-charge interaction with the amine moiety of the ligand (Fig. 2). The transmembrane segment amino acids that form the QNB binding pocket are identical in all five muscarinic receptor subtypes (Supplementary Table 1), consistent with results of QNB binding experiments on M1–M4 receptors, and with site-directed mutagenesis experiments on M119, M220, and M321 receptors. Only Phe181, which extends downward from ECL2 and interacts with one of the two phenyl rings on QNB (Fig. 2), differs from all other muscarinic receptor subtypes which have leucine in the homologous position. The importance of Asp3.32 for both agonist and antagonist binding has been demonstrated in mutagenesis and covalent-labeling experiments and modeling studies19–22. In contrast, mutation of Asn4046.52 to Ala on M123 and M324 receptors was shown to greatly affect binding of QNB but have little effect on binding of or activation by acetylcholine. It is possible that Asn4046.52 is hydrogen-bonded with ester group of QNB but not of acetylcholine.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Faculty of Science, Gakushuin University, Mejiro 1-5-1, Tokyo 171-8588, Japan.

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.

Show MeSH
Related in: MedlinePlus