The structural study of mutation-induced inactivation of human muscarinic receptor M4

IUCrJ. 2020 Feb 22;7(Pt 2):294-305. doi: 10.1107/S2052252520000597. eCollection 2020 Mar 1.


Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer's disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson's disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N4497.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination.

Keywords: Alzheimer’s disease; G-protein-coupled receptors; GPCRs; M4; Parkinson’s disease; ligand screening; muscarinic acetylcholine receptors; mutation design.

Grant support

This work was funded by National Natural Science Foundation of China grants 31870744 and 31971178. ShanghaiTech University grant .