Cryo-EM reveals an extrahelical allosteric binding site at the M5 mAChR

Wessel A. C. Burger, Jesse I. Mobbs, Bhavika Rana, Jinan Wang, Keya Joshi, Patrick R. Gentry, Mahmuda Yeasmin, Hariprasad Venugopal, Aaron M. Bender, Craig W. Lindsley, Yinglong Miao (), Arthur Christopoulos (), Celine Valant () and David M. Thal ()
Additional contact information
Wessel A. C. Burger: Monash University
Jesse I. Mobbs: Monash University
Bhavika Rana: Monash University
Jinan Wang: University of North Carolina
Keya Joshi: University of North Carolina
Patrick R. Gentry: Monash University
Mahmuda Yeasmin: Monash University
Hariprasad Venugopal: Monash University
Aaron M. Bender: Vanderbilt University
Craig W. Lindsley: Vanderbilt University
Yinglong Miao: University of North Carolina
Arthur Christopoulos: Monash University
Celine Valant: Monash University
David M. Thal: Monash University

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: Abstract The M5 muscarinic acetylcholine receptor (M5 mAChR) represents a promising therapeutic target for neurological disorders. However, the high conservation of its orthosteric binding site poses 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 remains limited. Here, we present a 2.8 Å 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 implement an integrated approach combining mutagenesis, pharmacological assays, structural biology, and molecular dynamics simulations. Our mutagenesis studies reveal that selective M5 PAMs bind outside previously characterized M5 mAChR allosteric sites. Subsequently, we obtain a 2.1 Å structure of M5 mAChR co-bound with acetylcholine and the selective PAM VU6007678, revealing an allosteric pocket at the extrahelical interface between transmembrane domains 3 and 4 that is 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 allosteric binding sites.

Date: 2025
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DOI: 10.1038/s41467-025-62212-z

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