Dynamics of the acetylcholinesterase tetramer

Abstract

Acetylcholinesterase rapidly hydrolyzes the neurotransmitter acetylcholine in cholinergic synapses, including the neuromuscular junction. The tetramer is the most important functional form of the enzyme. Two low-resolution crystal structures have been solved. One is compact with two of its four peripheral anionic sites (PAS) sterically blocked by complementary subunits. The other is a loose tetramer with all four subunits accessible to solvent. These structures lacked the C-terminal amphipathic t-peptide (WAT domain) that interacts with the proline-rich attachment domain (PRAD). A complete tetramer model (AChEt) was built based on the structure of the PRAD/WAT complex and the compact tetramer. Normal mode analysis suggested that AChEt could exist in several conformations with subunits fluctuating relative to one another. Here, a multiscale simulation involving all-atom molecular dynamics and C alpha-based coarse-grained Brownian dynamics simulations was carried out to investigate the large-scale intersubunit dynamics in AChEt. We sampled the ns-mus timescale motions and found that the tetramer indeed constitutes a dynamic assembly of monomers. The intersubunit fluctuation is correlated with the occlusion of the PAS. Such motions of the subunits "gate" ligand-protein association. The gates are open more than 80% of the time on average, which suggests a small reduction in ligand-protein binding. Despite the limitations in the starting model and approximations inherent in coarse graining, these results are consistent with experiments which suggest that binding of a substrate to the PAS is only somewhat hindered by the association of the subunits.

FIGURE 1

Model-built AChEt structure (left) and its schematic representation (right) showing subunits A (black), B (red), C (green), and D (blue). The ColQ onto which the WAT domains (residues 544–583) wrap around is in purple. Note the nearly symmetric arrangement of the monomers in this model. The three PAS residues (Tyr⁷², Trp²⁸⁶, and Tyr³⁴¹) are shown in yellow vdW spheres. The distances (d) between centers of subunits, the angle (θ) formed by three centers of subunits, and interfacial contact (χ) are illustrated. Note that χ represents the intersection (yellow sphere) of two selections: the numbers of atoms in one subunit that are within 10 Å of any atom in a complementary subunit and vice versa (see green and lavender arcs).

FIGURE 2

Time evolution of AChEt catalytic subunits' (residues 5–543, excluding the WAT domain) structural properties during the 20 ns AA-MD (left) and one of the (CG-BD, right) simulations. (A) RMSD and (B) RMSF in Å calculated after individually fitting the Cα atoms of each catalytic subunit. Color code: A (black), B (red), C (green), and D (blue).

FIGURE 3

BNM (left) and PCA (right). The BNMs were calculated for all three snapshots used for starting the CG-BD runs as well as for the last snapshot; a representative result from a snapshot at 7.72 ns, which was used to start run II, is shown here. Similarly, the PCA shown here is only for run II. The rest of the BNM and PCA calculations are very similar. (A–C) Representative modes from the first, second, and third sets of lowest frequency BNMs (see text) and the first, second, and third PCs. The blue arrows, drawn every four residue for clarity, indicate the direction of motion. For each mode (i.e., for those in A, B, and C), the same orientation is used and for different modes (i.e., A versus B, etc.), structures are rotated for the best view.

FIGURE 4

Distance (d), angle (θ), number of interfacial contacts (χ), and occlusion involving the catalytic domains (residues 5–543). (A) d between the center-of-mass of two subunits: A-B (black), B-C (red), C-D (green), and D-A (blue). (B) θ subtended by the centers of three subunits: (black), (red), (green), and (blue). (C) χ defined as the mean of the numbers of atoms in subunit i within 10 Å of any atom of subunit j and vice versa: A-B (black), B-C (red), C-D (green), and D-A (blue). (D) Occlusion defined as the number of atoms in a complementary subunit j within 16 Å of the PAS residues 72, 286, and 341 of subunit i: A (black), B (red), C (green), and D (blue).

FIGURE 5

Histograms of the deviations from planarity, defined as where θ is as defined in the legend of Fig. 4 and k is an index for the angles. The magenta dashed line represents the nonplanarity of the x-ray structure 1C2O.

FIGURE 6

Plots of occlusion against the intersubunit distance (main plot) and interfacial contact (inset) for the A-D pair. The correlation coefficients are indicated. A similar behavior was obtained for the other subunit pairs.

FIGURE 7

Statistical analysis of the PAS occlusion. (A) Probability distribution of the occlusion in one of the CG-simulations. Vertical dashed lines indicate the cutoffs used to define three states represented by the snapshots having an occlusion less than or equal to 2 (state 1), between 2 and 5 (state 2), or greater than 5 (state 3). (B) The fraction of structures in state 1 (square) and state 2 (circle) for subunits A, B, C, and D derived from each of the four CG-BD runs.

FIGURE 8

Docking of ACh into the PAS of AChE. (A) Docking results. Two hundred ACh structures were docked into three of the four PASs representing the open (occlusion = 0), partially occluded (occlusion = 2), and occluded (occlusion = 6) states. See text and legends of Figs. 1 and 5 for the definitions of PAS and occlusion. (B) A zoom-in on the PAS residues and the ACh structures docked onto PAS.