Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor


Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand–residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.

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Fig. 1: Structures of M2-BRIL and the S110R mutant bound to NMS.
Fig. 2: Binding assay of M2 variants with agonist and antagonists.
Fig. 3: Conformational changes of the tyrosine lid.
Fig. 4: Binding mode of the M2 receptor to AF-DX 384.
Fig. 5: Analysis of structural stabilization of the S110R M2 mutant compared to the wild-type M2 using MD simulations.
Fig. 6: Analysis of subtype selectivity of AF-DX 384 using MD simulation.

Data availability

The atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession codes 5ZK8 (M2-BRIL–NMS), 5ZKC (S110R-BRIL–NMS), 5ZKB (S110R-BRIL–AF-DX 384), 5ZK3 (S110R-BRIL–QNB), 5YC8 (S110R-BRIL–NMS:Hg). Raw diffraction images have been also deposited in Zenodo data repository ( for S110R-BRIL–NMS:Hg, for others).


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We acknowledge support from the Research Acceleration Program of the JST (S.I.); the Platform Project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT); the JSPS-NSF International Collaboration in Chemistry (ICC) (T.K. and B.K.); the Takeda Science Foundation (T.K. (Kyoto University) and R.S.); the Japan Agency for Medical Research and Development (AMED) (T.K. and T.M.); JSPS KAKENHI (Grant No. 15K08268 to R.S.; 15H06862 to K.Y.); and the ImPACT Program of the Council for Science, Technology and Innovation (Cabinet Office, Government of Japan) (T.M.). The MD simulations performed by S.L. and N.V. were supported by NIH R01-GM097261 (N.V.). We thank the beamline staff at SPring-8 for data collection and processing, and T. Sumiyoshi for providing the information about the ligands. The X-ray crystallography data collection was performed at SPring-8 (Proposal No. 2013A1379, 2013B1092, 2013B1184, 2014A1301, 2014B1273, 2014B1355, 2015A1044, 2015A1080, 2015B2044, and 2015B2080). DNA sequencing analysis was performed at the Medical Research Support Center, Graduate School of Medicine, Kyoto University. T. Nakagita made a diagram of the structure formula of ligands.

Author information




R.S. and T.K. designed the project. S.Y., T.M., and M.K. discovered the thermostabilizing mutant using the theoretical strategy. R.S., M.S.T., and H.T. carried out expression and purification of the receptor. H.T and S.M. carried out the binding assay. R.S. and M.S.T. crystallized the receptor. R.S., K.Y., and K.H. collected and processed the diffraction data. M.Y. supervised the data collection and data processing. R.S. and S.H solved and refined the structures. S.L. carried out MD simulations. S.L. and N.V. performed analysis and N.V. wrote the manuscript associated with the MD simulations. T.K, B.K.K., and S.I. supervised the overall project. R.S., S.M., N.V. and T.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Ryoji Suno or Takuya Kobayashi.

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Suno, R., Lee, S., Maeda, S. et al. Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor. Nat Chem Biol 14, 1150–1158 (2018).

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