Acetylcholinesterase: from 3D structure to function

Abstract

By rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase terminates neurotransmission at cholinergic synapses. Acetylcholinesterase is a very fast enzyme, functioning at a rate approaching that of a diffusion-controlled reaction. The powerful toxicity of organophosphate poisons is attributed primarily to their potent inhibition of acetylcholinesterase. Acetylcholinesterase inhibitors are utilized in the treatment of various neurological disorders, and are the principal drugs approved thus far by the FDA for management of Alzheimer's disease. Many organophosphates and carbamates serve as potent insecticides, by selectively inhibiting insect acetylcholinesterase. The determination of the crystal structure of Torpedo californica acetylcholinesterase permitted visualization, for the first time, at atomic resolution, of a binding pocket for acetylcholine. It also allowed identification of the active site of acetylcholinesterase, which, unexpectedly, is located at the bottom of a deep gorge lined largely by aromatic residues. The crystal structure of recombinant human acetylcholinesterase in its apo-state is similar in its overall features to that of the Torpedo enzyme; however, the unique crystal packing reveals a novel peptide sequence which blocks access to the active-site gorge.

Figure 1

Enzymatic hydrolysis of ACh by AChE.

Figure 2

Schematic representation of the binding sites of AChE based upon biochemical studies performed prior to determination of the 3D structure. ES, esteratic site; AS, anionic substrate binding site; ACS, aromatic cation binding site; PAS, peripheral anionic binding site. The hatched areas represent putative hydrophobic binding regions. ACh is shown spanning the esteratic and anionic sites of the catalytic center. Imidazole and hydroxyl side chains of His and Ser are shown within the esteratic site. Within the anionic site, (COO⁻)_n represents 6–9 putative negative charges.

Figure 3

Schematic representation of the GPI-anchored TcAChE dimer. The red lightning bolts indicate the site of hydrolysis by a bacterial PI-specific phospholipase C [36].

Figure 4

3D structure of TcAChE displayed as a ribbon diagram. The 14 conserved aromatic residues are shown as pink sticks and a dot surface. A model of the substrate, ACh, bound in the active site, is shown at the bottom of the active-site gorge, in ball-and-stick format.

Figure 5

Cartoon representation of the TcAChE dimer, with one monomer colored green and the other red. (a) View along the 2-fold axis; (b) View down the 2-fold axis. This type of dimer, held together by a 4-helix bundle as observed in the TcAChE structure [30], is virtually identical to that seen in the mouse [31] and Drosophila [32] AChE structures, as well as in the structures of human [33] and mouse [35] AChE complexed with the snake venom toxin, fasciculin.

Figure 6

Schematic diagram of the topological secondary structure of the α/β hydrolase fold. The α-helices are shown as red cylinders, the β-strands as turquoise arrows, and the three residues making up the active site are shown as green circles (the labels of Ser, Glu and His correspond to the catalytic-triad residues found in the AChE active-site).

Figure 7

Schematic view of the active-site gorge of TcAChE. The bottom of the gorge is characterized by several sub-sites: the anionic site, with which the choline moiety of ACh interacts; the esteratic site, which contains the three residues of the catalytic triad; the oxyanion hole, and the acyl pocket, which confers substrate specificity. The PAS is located ~15 Å above the active site, close to the mouth of the gorge.

Figure 8

The active site of AChE. (a) Model of ACh bound in the active site of TcAChE; (b) Close-up of the active site of the TMTFA-TcAChE complex [62], showing the experimentally determined TMTFA moiety (open-face lines) together with a superimposed model of ACh docked in the active site (solid lines). Several key residues in the binding pocket are indicated.

Figure 8

The active site of AChE. (a) Model of ACh bound in the active site of TcAChE; (b) Close-up of the active site of the TMTFA-TcAChE complex [62], showing the experimentally determined TMTFA moiety (open-face lines) together with a superimposed model of ACh docked in the active site (solid lines). Several key residues in the binding pocket are indicated.

Figure 9

Backbone drawings of TcAChE (left) and GLIP (right) with electrostatic potentials superimposed. Backbones of the proteins are displayed as white “worms”. The isopotential surfaces were generated using the program GRASP [79]. The red surface corresponds to the isopotential contour, −1kT/e, and the blue one to the isopotential contour, +1kT/e, where k is the Boltzmann constant, T is the temperature, and e is the electronic charge. Arrows indicate the direction of the dipole in each protein (taken from Ref. [75]).

Figure 10

Structural kinetics of covalent modification of TcAChE by the nerve agent, VX, as monitored by X-ray crystallography. The active site of TcAChE is depicted with possible H-bonds involving the catalytic triad and the OP moiety (broken lines). (a) Native structure, showing the active site, including the catalytic triad (S200-H440-E327) and the oxyanion hole (-NH of G118, G119, and A201); (b) Pro-aged structure. Phosphonylation triggers a conformational change of H440 that disrupts the H-bond to G327; this may be caused either by steric crowding in the pentavalent phosphorus transition state, or by re-distribution of charge on the H440 imidazole during phosphonylation. It should be noted that E199 and a water molecule apparently stabilize the alternate conformation of H440. Subsequently, the H440 imidazole catalyzes either dealkylation (aging), or spontaneous reactivation; (c) Aged structure. For reaction of AChE with VX and with most phosphonates, aging predominates, and dealkylation results in movement of H440 into the negatively charged pocket formed by E327-Oε, S200-Oγ, and one anionic oxygen of the dealkylated OP.

Figure 10

Structural kinetics of covalent modification of TcAChE by the nerve agent, VX, as monitored by X-ray crystallography. The active site of TcAChE is depicted with possible H-bonds involving the catalytic triad and the OP moiety (broken lines). (a) Native structure, showing the active site, including the catalytic triad (S200-H440-E327) and the oxyanion hole (-NH of G118, G119, and A201); (b) Pro-aged structure. Phosphonylation triggers a conformational change of H440 that disrupts the H-bond to G327; this may be caused either by steric crowding in the pentavalent phosphorus transition state, or by re-distribution of charge on the H440 imidazole during phosphonylation. It should be noted that E199 and a water molecule apparently stabilize the alternate conformation of H440. Subsequently, the H440 imidazole catalyzes either dealkylation (aging), or spontaneous reactivation; (c) Aged structure. For reaction of AChE with VX and with most phosphonates, aging predominates, and dealkylation results in movement of H440 into the negatively charged pocket formed by E327-Oε, S200-Oγ, and one anionic oxygen of the dealkylated OP.

Figure 10

Structural kinetics of covalent modification of TcAChE by the nerve agent, VX, as monitored by X-ray crystallography. The active site of TcAChE is depicted with possible H-bonds involving the catalytic triad and the OP moiety (broken lines). (a) Native structure, showing the active site, including the catalytic triad (S200-H440-E327) and the oxyanion hole (-NH of G118, G119, and A201); (b) Pro-aged structure. Phosphonylation triggers a conformational change of H440 that disrupts the H-bond to G327; this may be caused either by steric crowding in the pentavalent phosphorus transition state, or by re-distribution of charge on the H440 imidazole during phosphonylation. It should be noted that E199 and a water molecule apparently stabilize the alternate conformation of H440. Subsequently, the H440 imidazole catalyzes either dealkylation (aging), or spontaneous reactivation; (c) Aged structure. For reaction of AChE with VX and with most phosphonates, aging predominates, and dealkylation results in movement of H440 into the negatively charged pocket formed by E327-Oε, S200-Oγ, and one anionic oxygen of the dealkylated OP.

Figure 11

Ribbon diagram of the overall [WAT]₄PRAD crystal structure. The structure reveals four WAT helices wrapped around a single PRAD helix. Color coding is from blue at the N-terminus to red at the C-terminus for each chain, showing that the four WATs run parallel to each other, while PRAD runs anti-parallel to all WAT chains. Note that each WAT helix has a different height; as a consequence, each of the four interacts with a different region of the PRAD.

Figure 12

Model of the physiological ColQ-linked AChE_T tetramer. The four catalytic subunits surround the ColQ polypeptide, with the WAT sequences displayed as ribbons, and the PRAD as a grey surface model. In this model, access to the active-site gorge (yellow patches) is from the top in two opposing catalytic subunits, while the other two open downwards, in the direction of the basal lamina, as described in [113].

Figure 13

Ribbon model of the dimeric structure of apo rhAChE. The view displayed is down the 2-fold NCS axis of the dimer in the ASU. The cysteine residues involved in disulfide bond formation are shown as balls (with green balls representing carbon atoms, blue balls nitrogen atoms, red balls oxygen atoms, and orange balls sulfur atoms).

Figure 14

Packing of two rhAChE dimers. Each dimer consists of two AChE catalytic subunits (A in orange and B in blue), which are related by the 2-fold NCS axis shown in Fig. 13. A surface loop (residues 489–499, green balls-and-sticks) of subunit A interacts with the PAS of subunit B from a symmetry-related dimer. Since the blue subunits have no adjacent PAS to interact with, their corresponding loops are disordered, as is the case in many other AChE structures.

Figure 15

The PAS of rhAChE interacts with the positively charged loop on the adjacent monomer. The adjacent monomer is rendered in grey surface format. Residues in the vicinity (<4.5 Å) of the binding site for the basic loop are highlighted on the surface in stick representation (with carbon atoms color-coded in yellow, oxygen atoms in red, and nitrogens atoms in blue). The refined N-acetylglucosamine (NAG) in the proximity of the interface is also shown.

Figure 16

Electron density around the active-site ‘blocking’ loop in the structure of apo rhAChE. The loop region (residues 490–498) is shown as green sticks with the refined 2Fo-Fc electron density around it contoured at 1.5 σ. Side-chains in the ‘blocked’ subunit within 5.5 Å of the loop are displayed as grey sticks. Key residues are marked for orientation.