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Xanomeline displays concomitant orthosteric and allosteric binding modes at the M₄ mAChR.

ABSTRACT: The M₄ muscarinic acetylcholine receptor (M₄ mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M₄ mAChR in complex with the heterotrimeric G_i1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M₄ mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M₄ mAChR. These findings provide a basis for further understanding xanomeline's complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.

SUBMITTER: Burger WAC

PROVIDER: S-EPMC10482975 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Xanomeline displays concomitant orthosteric and allosteric binding modes at the M4 mAChR.

Burger Wessel A C WAC Pham Vi V Vuckovic Ziva Z Powers Alexander S AS Mobbs Jesse I JI Laloudakis Yianni Y Glukhova Alisa A Wootten Denise D Tobin Andrew B AB Sexton Patrick M PM Paul Steven M SM Felder Christian C CC Danev Radostin R Dror Ron O RO Christopoulos Arthur A Valant Celine C Thal David M DM

Nature communications 20230906 1

The M4 muscarinic acetylcholine receptor (M4 mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM ...[more]

PMID: 37673901

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Project description:Apolipoprotein-D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein-D adopts an eight-stranded β-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α-helix. Crystallography studies of recombinant apolipoprotein-D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX-MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X-ray crystallography, making HDX-MS an invaluable orthogonal technique. Here, we report an HDX-MS protocol for apolipoprotein-D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand-free apolipoprotein-D revealed apolipoprotein-D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein-D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N-terminus and C-terminus. Together, our experiments provide insight into apolipoprotein-D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein-D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein-D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein-D structure from X-ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein-D, to elucidate how proteins change flexibility when binding to small molecules.

Dataset Information

Xanomeline displays concomitant orthosteric and allosteric binding modes at the M₄ mAChR.

Publications

Xanomeline displays concomitant orthosteric and allosteric binding modes at the M<sub>4</sub> mAChR.

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