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Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

Keov, P; Lopez, L; Devine, S M; Valant, C; Lane, J R; Scammells, P J; Sexton, P M; Christopoulos, A


P Keov

L Lopez

S M Devine

C Valant

Associate Professor

P J Scammells

P M Sexton

A Christopoulos


TBPB and 77-LH-28-1 are selective agonists of the M1 muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M1 mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.


Keov, P., Lopez, L., Devine, S. M., Valant, C., Lane, J. R., Scammells, P. J., …Christopoulos, A. (2014). Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor. Journal of Biological Chemistry, 289(34), 23817-23837.

Journal Article Type Article
Acceptance Date Jul 8, 2014
Online Publication Date Jul 8, 2014
Publication Date Aug 22, 2014
Deposit Date Apr 22, 2020
Journal Journal of Biological Chemistry
Publisher American Society for Biochemistry and Molecular Biology
Peer Reviewed Peer Reviewed
Volume 289
Issue 34
Pages 23817-23837
Public URL
Publisher URL