Gentry, P. R. | bioRxiv Science

bioRxiv2025

Cryo-EM reveals a new allosteric binding site at the M5 mAChR

The M5 muscarinic acetylcholine receptor (M5 mAChR) represents a promising therapeutic target for neurological disorders. However, the high conservation of its orthosteric binding site has posed significant challenges for drug development. While selective positive allosteric modulators (PAMs) offer a potential solution, a structural understanding of the M5 mAChR and its allosteric binding sites has remained limited. Here, we present a 2.8 [A] cryo-electron microscopy structure of the M5 mAChR complexed with heterotrimeric Gq protein and the agonist iperoxo, completing the active-state structural characterization of the mAChR family. To identify the binding site of M5-selective PAMs, we implemented an integrated approach combining mutagenesis, pharmacological assays, structural biology, and molecular dynamics simulations. Our mutagenesis studies revealed that selective M5 PAMs bind outside previously characterized M5 mAChR allosteric sites. Subsequently, we obtained a 2.1 [A] structure of M5 mAChR co-bound with acetylcholine and the selective PAM VU6007678, revealing a novel allosteric pocket at the extrahelical interface between transmembrane domains 3 and 4 that was confirmed through mutagenesis and simulations. These findings demonstrate the diverse mechanisms of allosteric regulation in mAChRs and highlight the value of integrating pharmacological and structural approaches to identify novel allosteric binding sites.

pharmacology and toxicology↗

bioRxiv2019

Crystal structure of the M5 muscarinic acetylcholine receptor

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all five mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies together with chimeric swaps between the extracellular regions of the M2 and M5 mAChR further revealed the structural insight into \"kinetic-selectivity\", where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective ligands.\n\nSignificance StatementThe five subtypes of the muscarinic acetylcholine receptors (mAChRs) are expressed throughout the central and peripheral nervous system where they play a vital role in physiology and pathologies. Recently, the M5 mAChR subtype has emerged as an exciting drug target for the treatment of drug addiction. We have determined the atomic structure of the M5 mAChR bound to the clinically used inverse agonist tiotropium. The M5 mAChR structure now allows for a full comparison of all five mAChR subtypes and reveals subtle differences in the extracellular loop (ECL) regions of the receptor that mediate orthosteric and allosteric ligand selectivity. Together these findings open the door for future structure-based design of selective drugs that target this therapeutically important class of receptors.

pharmacology and toxicology↗