A single molecular distance predicts agonist binding energy in nicotinic receptors

Abstract

Agonists turn on receptors because they bind more strongly to active (R*) versus resting (R) conformations of their target sites. Here, to explore how agonists activate neuromuscular acetylcholine receptors, we built homology models of R and R* neurotransmitter binding sites, docked ligands to those sites, ran molecular dynamics simulations to relax ("equilibrate") the structures, measured binding site structural parameters, and correlated them with experimental agonist binding energies. Each binding pocket is a pyramid formed by five aromatic amino acids and covered partially by loop C. We found that in R* versus R, loop C is displaced outward, the pocket is smaller and skewed, the agonist orientation is reversed, and a key nitrogen atom in the agonist is closer to the pocket center (distance d_x) and a tryptophan pair but farther from αY190. Of these differences, the change in d_x shows the largest correlation with experimental binding energy and provides a good estimate of agonist affinity, efficacy, and efficiency. Indeed, concentration-response curves can be calculated from just d_x values. The contraction and twist of the binding pocket upon activation resemble gating rearrangements of the extracellular domain of related receptors at a smaller scale.

Figure 1.

Sites, agonists, and cycle. (a) Ligand binding sites. Side view of an acetylcholine binding protein, homologous to the AChR extracellular domain (PDB ID: 3WIP; Olsen et al., 2014). Sites are at subunit interfaces. The agonist (ACh, yellow) is surrounded by a cluster of aromatic residues (green). In endplate AChRs, the principal subunit (white) is α and the complementary subunit is δ, ε (adult), or γ (fetal). Bottom right: Top-view schematic of endplate AChR subunits (neurotransmitter binding sites, red). (b) Agonist structures. The principal nitrogen is blue. (c) Cyclic activation scheme for a receptor having one functional binding site. Horizontal, agonist binding and vertical, receptor activation (“gating”). R, resting state (low affinity and closed channel); R*, active state (high affinity and open channel); A, agonist. Boxed, equilibrium constants and free-energy changes (in the direction of the arrow). K_dR and K_dR*, resting and active equilibrium dissociation constants; E₀ and E₁, un- and mono-liganded gating equilibrium constants.

Figure 2.

Binding site structures (x-ray). (a) AChBP with ACh (top; PDB ID: 3WIP; Olsen et al., 2014) or nicotine (bottom; PDB ID: 1UW6; Celie et al., 2004). Dashed lines are H-bonds; red dot is a structural water. (b) α4β2 AChR with nicotine (top; PDB ID: 5KXI; Morales-Perez et al., 2016) or docked ACh (bottom). In both structures, before equilibration the agonist tail points toward the complementary subunit, but after equilibration it is flipped only in α4β2 (Table 1).

Figure 3.

Binding site structural parameters. Pocket volume was calculated as that of the pyramid formed by joining the centers of the five aromatic rings (front face, white). Distances are between the agonist’s principal nitrogen (Fig. 1 b) and the pocket center (d_x) or the five ring centers (d₉₃, d₁₄₉, d_190, d₁₉₈, and d₅₅), and between loop C and the complementary subunit backbone (d_loopC). Angle Θ_a is the agonist’s orientation. Not shown: angles Θ_s (pocket skew), Θ_W (between indole planes), Θ_p (between pyramid and pore axes), and density of water in the pocket. PDB ID: 3WIP; residue numbers are for endplate AChRs.

Figure 4.

RMSD of the protein backbone. The backbone equilibrates within ∼10 ns. Most of the residual fluctuations are from loop F.

Figure 5.

Pocket volume and d_x. Resting, brown and active, green. (a) Top: Pocket volume. With all agonists, α−γ is the smallest (^AR and ^AR*). At all sites, ^AR* is smaller than ^AR (all agonists) and pocket volume is smallest with TMA. Bottom: d_x. Distance between the agonist’s principal nitrogen and the pocket center is approximately twofold smaller in ^AR* versus ^AR (all sites and agonists). (b) Example d_x distributions. Each panel shows results from two MD trajectories (α−γ).

Figure 6.

Water density. The number of water molecules was counted in spheres of radii 5, 10, and 20 Å, with the origin at the pocket center (α−γ). There is no significant difference between ^AR and ^AR* conformations or between agonists. The 20 Å values are the same as for the bulk solution (dashed line, 1 molecule per ų or 29.8 g/cm³).

Figure 7.

Resting versus active α−γ pocket metrics. Distances are ^AR*/^AR ratios and angles are ^AR* – ^AR differences. Left and center columns: Metrics that differ significantly between ^AR* and ^AR. For ACh-class agonists (colored bars), in R* pocket volume is ∼40% smaller and 1/d_x is ∼50% smaller. For Epx (gray), in R* pocket volume is 22% smaller and 1/d_x is 37% smaller. Right column: Metrics that are the same in R and R*.

Figure 8.

Energy–structure correlations. (a) Linear correlations between experimental binding energy and binding site metrics (ACh, CCh, TMA, and Cho; all sites; ^AR and ^AR*). The highest correlation is with d_x. (b) Pearson’s correlation coefficients by agonist (dashed lines, P < 0.0001 significance threshold).

Figure 9.

Resting versus active α−γ neurotransmitter binding site. (a) For all agonists, the active pocket (^AR*, green) is smaller than the resting pocket (^AR, brown; Fig. 3). (b) ^AR* versus ^AR with ACh. In the active state, the agonist’s quaternary nitrogen (blue dot) is closer to the pocket center (black dot) and the agonist’s tail (red arrow) points away from the complementary subunit. (c) With ACh, in ^AR* versus ^AR, the tryptophan pair is closer to and αY190 is further from the quaternary nitrogen of ACh (blue dot). (d) In ^AR* versus ^AR, loop C in the α subunit is displaced outward, but loop E in the γ subunit is the same. The pore axis is approximately vertical.

Figure 10.

Affinity, efficacy, and efficiency. In each plot, y-axis values are free energies from electrophysiology experiments and x-axis values are distances from equilibrated structures. Open symbols, ^AR; closed symbols, ^AR*. (a) Distance between the agonist’s principal nitrogen (Fig. 1 b) and the pocket center (Fig. 3) is correlated linearly with agonist binding energy (Eq. 6; slope = 26.3 ± 1.6 Å · kcal/mol; y-intercept = 2.8 ± 0.6 kcal/mol). (b) Relative efficacy correlates with the active–resting difference in 1/d_x. (c) The d_x ratio, ^AR*/^AR, predicts energy efficiency (within ∼10%). The higher efficiency of α−γ versus α-δ/ε (lines mark means) and the lower efficiency of Epx are apparent.

Figure 11.

CRCs. Symbols are from electrophysiology experiments, and solid lines are calculated from d_x values. Inset: CRCs for two agonists that were not used in the energy–d_x correlation (Fig. 10 a). See Fig. 1 b for agonist structures.