Figure 1.
Schematic of neuronal nAChR structure.
The proportions of the extracellular domain (A), transmembrane domain (B), and intracellular domain (C) are illustrated on the right while the modeled subunit stoichiometry and configuration for heteromeric neuronal nAChRs is illustrated on the left, including labels for the (+) and (−) side of each subunit and the location of each of the two agonist binding sites.
Figure 2.
Compounds used in hα3β4 and hα4β2 nAChR ECD blind docking.
A. Docked agonists included acetylcholine, nicotine, and epibatidine. B. Docked antagonists included COB-3, PPB-9, APB-12, and KAB-18.
Figure 3.
Histograms of model energies per modeling iteration.
The number of models per cluster were plotted against the total calculated model energy (internal energy + PB solvation energy term) in kcal/mol * 10−3. Iterations 2-7 incorporates the top scoring model from the previous iteration as an additional template. 1. Two roughly aligned AChBP templates (PDB ID: 1UWG and PDB ID: 2BYR) were used with symmetry restraints. 2. An additional AChBP template (PDB ID: 2BJ0) was included; template alignment was refined, secondary structure assignments and distance restraints of select conserved motifs were added. 3. β-sheet restraints were added. 4. Mouse α1 monomer (PDB ID: 2QC1) was included as a fourth crystallographic template; α1 template specifically used to refine loop 1; hydration pocket waters added. 5. α1 template was used to refine F loop conformation. 6. C loop conformation of β subunits was refined. 7. The A loop of all subunits were refined with a template modified by LES MD simulation; symmetry were restraints removed. 8. Human α3β4 ECD models built using same alignments and constraints as in G. 9. Human α4β2 ECD models built using same alignments and constraints as in G.
Figure 4.
Blind docking modes compared to X-ray structures.
Docking modes for epibatidine (A – magenta), nicotine (B – orange), and acetylcholine (C – green) to hα4β2 models compared to crystallographic binding modes (blue). Crystallographic structures for AChBP bound to epibatidine, nicotine, and carbamylcholine (PDB IDs: 2BYN, 1UW6, and 1UV6 respectively) were superimposed on nAChR ECD models to determine RMSDs of the dockings.
Table 1.
Blind docking results for agonists to multiple hα4β2 and hα3β4 nAChR ECD conformations.
Figure 5.
Detailed antagonist docking modes.
A. Docking mode of KAB-18 (magenta) at the α4(+) (green)/β2(-) (blue) interface in the presence of the agonist epibatidine (grey). Residues varying between the β2 and β4 subunits are featured (dark blue). B. Superimposed Glide docking modes of KAB-18 (magenta), APB-12 (cyan), PPB-9 (orange), and COB-3 (green) at the same binding site.
Table 2.
Measurements of agonist binding distances in MD simulations of epibatidine bound hα4β2 and hα3β4 nAChR ECDs.
Figure 6.
C loop closure of AChBP bound to various ligands.
Superposition of four crystal structures of AChBP in complex with various compounds to illustrate the difference in intersubunit distances between Cα of residue C191 of the α subunit C loop on the (+) side of the binding interface and Cα of residue 58 of the β subunit β2 strand on the (−) side of the interface. Only epibatidine is shown (pink surface) for clarity, to highlight the ligand binding site. The tabulated Cα-Cα distances allows for quantification of the degree of C loop closure upon ligand binding.
Table 3.
General ranges for C loop “openness” upon binding ligands of different pharmacological function measured from AChBP X-ray structures.
Table 4.
Measurements of C loop closure for MD simulations of epibatidine bound hα4β2 and hα3β4 nAChR ECDs.
Table 5.
MM-PBSA binding energy calculations for epibatidine and KAB-18 bound to hα4β2 and hα3β4 nAChR ECD models.
Table 6.
Effects of agonists and antagonists on wild-type and mutated human α4β2 nAChRs.
Figure 7.
Comparison of experimental DMXBA binding to computationally predicted KAB-18/epibatidine binding.
The X-ray structure of DMXBA (orange) in complex with Aplysia californica AChBP (grey ribbon) superimposed on a hα4β2 nAChR ECD model (green and blue ribbon for α4 and β2 subunits, respectively) bound to both epibatidine (grey) and the negative allosteric modulator KAB-18 (magenta). The C loops for each protein have been removed for clarity in the main figure, while the inset features the varied degree of C loop closure.