Structure and Function Prediction of Human Muscarinic Acetylcholine Receptor 1, Cation-π Studies, and Protein Design

Citation

Peng, Joyce Yaochun (2005) Structure and Function Prediction of Human Muscarinic Acetylcholine Receptor 1, Cation-π Studies, and Protein Design. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/XVJR-RN32. https://resolver.caltech.edu/CaltechETD:etd-05312005-114949

Abstract

(1) Muscarinic acetylcholine receptors, a G protein-coupled receptor, are responsible for a wide range of diseases. We predicted the 3D structure of the human M1 muscarinic receptor using the MembStruk method and validated its binding sites for 10 agonists and antagonists using the HierDock method. The predicted binding sites, the intramolecular contacts that stabilize the receptor conformation, and the in silico mutagenesis results, agree well with mutagenesis data. The calculated relative binding energies correlate well with measured binding affinities. In addition, the predicted binding sites provide a structural basis for the large reduction in ligand binding affinity and signaling efficacy by Trp 157 and Pro 159 mutations, which was not previously explained by homology models. The predicted binding sites illustrate the importance of aromatic residues in ligand binding through extensive cation-pi and aromatic-aromatic interactions, with new mutation candidates suggested. The predicted M1 structure improves our understanding of the muscarinic receptors, offers a basis for structure based drug design, and is a successful step toward applying these procedures in predicting the structures of other muscarinic receptor subtypes.

(2) We used high-level quantum mechanics to quantify cation-pi interactions in the crystal structure of carbamylcholine binding to Acetylcholine-binding Protein, a nicotinic receptor homolog. The calculated effects of fluorinated unnatural amino acid substitutions also correlate excellently with experimental EC50 data, suggesting that quantum mechanics can accurately predict cation-pi binding in a protein environment and provides a good model system in developing force fields to better describe cation-pi interactions.

(3) Histidines are known to modulate pH responsive binding. We designed a series of histidine derivatives by substituting its imidazole ring with functional groups that are small in size and lack the ability to form hydrogen bonds. Quantum mechanical calculations of the acid dissociation constants (pKa) show that these substitutions shift the histidine pKa upward or downward. We report a list of histidine derivatives and their corresponding pKa values that can be used in designing tumor specific drugs (e.g. HER2-Herceptin antibody), drug delivery through pH sensitive hydrogels, drug recycling, catalysis, and biosensors development. An example of how these unnatural histidines can be used is illustrated with 2-methyl histidine incorporated in a c-Myc-Max heterodimer.

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