On homology modeling of the M₂ muscarinic acetylcholine receptor subtype

Abstract

Twelve homology models of the human M2 muscarinic receptor using different sets of templates have been designed using the Prime program or the modeller program and compared to crystallographic structure (PDB:3UON). The best models were obtained using single template of the closest published structure, the M3 muscarinic receptor (PDB:4DAJ). Adding more (structurally distant) templates led to worse models. Data document a key role of the template in homology modeling. The models differ substantially. The quality checks built into the programs do not correlate with the RMSDs to the crystallographic structure and cannot be used to select the best model. Re-docking of the antagonists present in crystallographic structure and relative binding energy estimation by calculating MM/GBSA in Prime and the binding energy function in YASARA suggested it could be possible to evaluate the quality of the orthosteric binding site based on the prediction of relative binding energies. Although estimation of relative binding energies distinguishes between relatively good and bad models it does not indicate the best one. On the other hand, visual inspection of the models for known features and knowledge-based analysis of the intramolecular interactions allows an experimenter to select overall best models manually.

Fig. 1

Alignments of templates to target structure. Alignment of templates for homology modeling labeled by their PDB entry code to the target sequence of the human M₂ muscarinic acetylcholine receptor. Stars denote conserved and dots consensual residues. Colors denote secondary structure: red—helix; white—coil; yellow—strand; green—turn. Secondary structure of the target was predicted by PsiPred (http://bioinf.cs.ucl.ac.uk/psipred/) and secondary structures of templates were taken from respective crystal structures. For orientation transmembrane (TM) helices, inner (i) and outer (o) loops are indicated

Fig. 2

Twelve homology models of the M₂ receptor superposed on the 3UON crystal structure. Homology models (color) of the M₂ receptor based on the templates listed in Table 1 are superposed using MUSTANG [27] implemented in YASARA on the crystal structure 3UON (gray). Orientation: extracellular site up, TM VI and TM VII front. Colors: purple—α-helix; yellow—β-sheet; cyan—turn; white—coil. RMSDs of the models to the target structure (3UON) are listed in Table 4

Fig. 3

Correlation of calculated binding energies and RMSD of redocked QNB to 3UON. Calculated YASARA binding energies (top), AutoDock energies (middle) and Prime MMGB/SA energies (bottom) of poses from QNB re-docking to 3UON are plotted against their RMSDs. Detail with poses of RMSD of ligand smaller than 3 Å is on left and all poses are on right. Correlation coefficients are indicated in the graphs

Fig. 4

Calculated binding energies of muscarinic antagonists docked to homology models. Calculated binding energies of the antagonists QNB (squares), NMQNB (downward triangles), NMS (circles) and atropine (upward triangles) in YASARA (y-axis) are plotted against the binding energies calculated in Prime MMGB/SA (x-axis). Good poses are black, and bad poses are red. Closed symbols denote the best poses

Fig. 4

Calculated binding energies of muscarinic antagonists docked to homology models. Calculated binding energies of the antagonists QNB (squares), NMQNB (downward triangles), NMS (circles) and atropine (upward triangles) in YASARA (y-axis) are plotted against the binding energies calculated in Prime MMGB/SA (x-axis). Good poses are black, and bad poses are red. Closed symbols denote the best poses

Fig. 5

The best QNB docking poses of 12 homology models superposed on the 3UON crystal structure. View from the extracellular site, TM II down, TM VI and TM VII up, of the best docking poses, according to the binding energy estimates (Fig. 4), of QNB (green carbons) and residues of the orthosteric binding site (color) superposed on the crystal structure of 3UON (gray). Colors: Cyan—carbon; red—oxygen; blue—nitrogen; white—hydrogen; yellow—hydrogen bonds. The residue labels correspond to the M₂ sequence. The calculated RMSDs of QNB and residues of the orthosteric binding site of the models to the target structure (3UON) are shown in Table 5