Paper Citing NAMD - Abstract
Keov, Peter; Lopez, Laura; Devine, Shane M.; Valant, Celine; Lane, J. Robert; Scammells, Peter J.; Sexton, Patrick M.; Christopoulos, Arthur
Molecular Mechanisms of Bitopic Ligand Engagement with the M-1 Muscarinic Acetylcholine Receptor
JOURNAL OF BIOLOGICAL CHEMISTRY, 289:23817-23837, AUG 22 2014
Background: Bitopic ligands bind concomitantly to orthosteric and allosteric receptor sites. Results: Residues affecting binding and biased signaling of the selective agonists TBPB and 77-LH-28-1 were identified at the M-1 muscarinic receptor. Conclusion: Novel bitopic ligand binding poses and mechanisms of receptor activation were identified. Significance: Understanding the basis of bitopic ligand mechanisms can enable the design of selective ligands. TBPB and 77-LH-28-1 are selective agonists of the M-1 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 M-1 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.
