Figure 1.
Nicotinic acetylcholine receptor structure and α-conotoxin ImI.
(A) Nicotinic acetylcholine receptors (nAChRs) are ligand gated-ion channels. Their structure is composed of a ligand-binding domain (red), a transmembrane domain (blue), and an intracellular domain (white). nAChRs are permeable to Na+ and K+ and, for some isoforms, Ca2+. The opening of the channel is triggered by acetylcholine or nicotine. One of the acetylcholine binding sites is indicated as a blue star. (B) α-conotoxin ImI comprises 12 residues and is C-terminally amidated (indicated by * in the sequence). The structure features a short α-helix and two disulfide bonds that link cysteines I-III and II-IV.
Figure 2.
Sequence and structure of α7-nAChR ligand-binding domain.
Sequence alignment of Homo sapiens α7-nAChR ligand-binding domain (LBD) (UniProtKB/SwissProt P36544), Mus musculus α1-nAChR LBD (PDB ID: 1pq1), and Aplysia californica AChBP (PDB ID: 1tg9), which is structurally analogous to nAChRs. Below the alignment, the secondary structure elements and acetylcholine binding sites are shown on the lowest energy three-dimensional model of the α7-nAChR nAChR LBD obtained by comparative modeling. Residues in the sequence alignment are numbered according to the α7-nAChR sequence. The conserved positions between the three sequences are on a dark green background, whereas the positions presenting amino acids shared by only two sequences are on a light green background. The secondary structure elements are the α-helix h1 and the β-strands β1-10. The LBD is a pentamer of five subunits. The acetylcholine binding sites, indicated by star symbols, are located at the interface between the subunits. These binding sites mainly comprise the C-loop from one subunit, which is designated as the principal subunit, and the beta strands β1, β2, β3, β5′ and β6 from another subunit, which is designated as the complementary subunit. The secondary structures of one subunit are highlighted in the side view, and the arrangement of the subunits and of the binding sites is shown on the top view. In the alignment, the residues of AChBP in contact with ImI in the crystal structure 2c9t are underlined in blue for positions in the principal subunit and in white for positions in the complementary subunit.
Figure 3.
Analysis of the stability of α7-nAChR over 10 ns molecular dynamic simulations in the apo (A,C,E) and ImI-bound (B,D,F) states.
β strand α carbon root-mean-square deviations (RMSD) of each of the subunits over the molecular dynamics simulations to the starting frame for the apo (A) and ImI-bond models (B). α carbon root-mean-square fluctuation (RMSF) of each subunit of the apo (C) and ImI-bond (D) models. Fluctuation of the distance between the sulfur atom of α7-C190 side chain and the α carbon of α7-Y32 in the apo (E) and ImI-bond (F) models. This distance characterizes the closure of the C-loop. The RMSD is calculated using Cα atoms in β strands. The RMSD and distances were averaged using a 16 ps window.
Figure 4.
Comparison of the binding site of AChBP/ImI complex (PDB ID: 2c9t), α7-nAChR/ImI complex (model, this work) and α1-nAChR/bungarotoxin complex (PDB ID: 2qc1).
In the α1-nAChR/bungarotoxin structure, only one subunit was crystallized, and the bungarotoxin is not shown. The model displaying α7-nAChR was obtained by a combination of comparative modeling and molecular dynamics, and the displayed conformation corresponds to energetically minimized frames after 10 ns of simulations. The C-loop, the principal subunit, and the complement subunit are indicated. In the three first panels and from left to right, the conformation of the C-loop increasingly reduces the volume of the binding site. The fourth panel, on the right, shows a superimposition of the AChBP and nAChR subunits, highlighting the different C-loop conformations between the model and the two experimental templates.
Figure 5.
Analysis of the binding mode of ImI to the α7-nAChR.
The structure of the binding pocket occupied by ImI after molecular dynamics simulation is displayed and positions discussed in the text are highlighted. The α7 principal subunit is in orange, the α7 complementary subunit is in pale yellow, and ImI is in violet. Nitrogens are in blue, oxygens are in red and sulfurs are in yellow.
Figure 6.
Superimposition of wild-type and mutated models.
The models of the mutants shown were refined using molecular dynamics and the conformations shown in this figure are the last frames of the molecular dynamics trajectories. The arrows highlight local conformational changes.
Figure 7.
Correlation between experimentally derived and calculated mutational energies of the ImI mutants in the minimization based approach MBA.
Mutational energies were computed using either molecular mechanics generalized Born (GB) surface area (MM-GB/SA) or molecular mechanics Poisson-Boltzmann (PB) surface area (MM-PB/SA) energy functions at 298 K. The mutated models were refined using MBA with either distance dependent dielectric constant minimization (DDDCM) or explicit water minimization (EWM). Experimental mutational energies (▵▵G Exp) were derived using the corresponding Kd values of ImI wild-type/(7-nAChR and ImI mutants/(7-nAChR [26]–[28].
Table 1.
Calculated and experimental mutational energies (kcal/mol) of ImI mutants.
Figure 8.
Correlation between experimentally derived and calculated mutational energies of ImI mutants in the molecular dynamics based approach MDBA.
Mutational energies were computed by using either molecular mechanics generalized Born (GB) surface area (MM-GB/SA) or molecular mechanics Poisson-Boltzmann (PB) surface area (MM-PB/SA) approaches at 298 K. In the MDBA five alternative position restraint strategies were employed: (i) all receptor atoms >6 Å from the conotoxin were restrained to their position; (ii) all receptor atoms >4.5 Å from the conotoxin were restrained to their position; (iii) all the atoms located >6 Å from the mutated residue were restrained to their position; (iv) all the atoms from the receptor were restrained to their position, and all the atoms from the conotoxin mutants were free to move; and (v) all residues were restrained to their position. Experimental mutational energies (ΔΔG Exp) were derived using the corresponding Kd values of ImI wild-type/α7-nAChR and ImI mutants/α7-nAChR [26]–[28].
Table 2.
Calculated and experimental mutational energies (kcal/mol) of the receptor mutants.
Table 3.
Decomposition of the binding free energy (kcal/mol) of ImI and mutants.
Figure 9.
Correlation between calculated mutational energy components of ImI mutants and experimentally derived mutational energies.
The explicit water minimization approach was employed to compute the Gibbs free energy (ΔΔG) using either molecular mechanics generalized Born (GB) surface area (MM-GB/SA) or molecular mechanics Poisson-Boltzmann (PB) surface area (MM-PB/SA) approaches at 298 K. The energies were decomposed into van der Waals (vdw), electrostatic (ele), surface area (SA, only shown for GB) and entropic components (not shown). Experimental mutational energies (ΔΔG Exp) were derived using the corresponding Kd values of ImI wild-type/α7-nAChR and ImI mutants/α7-nAChR [26]–[28].
Table 4.
Decomposition of the mutational energies (kcal/mol) of ImI mutants.