Human Acetylcholinesterase
The human acetylcholinesterase (AChE) is an enzyme which hydrolyses the neurotransmitter acetylcholine (ACh) in the neuromuscular junctions and in other cholinergic synapses to terminate the neuronal signal. It has an ellipsoidal shape with dimensions ~ 45Å x 60Å x 65Å. This protein is composed of 531 residues. It consists of 12-stranded, central mixed β-sheet surrounded by 14 α helices. It is a member of the α/β hydrolase fold family[1]. In the physiological conditions, AChE exists as tetramers associated with either collagen-like Q subunit (ColQ) or proline-rich membrane-anchoring protein (PRiMA). The AChE is linked with these anchoring molecules by a "tryptophan amphiphilic tetramerization" domain (WAT). There is also a monomeric form which is soluble in the blood. ContentsThe Active site gorge of AChEThe active site of AChE involves two sites: the peripheral site and the catalytic site.
The peripheral site is a transitional binding site of the substrate. It provides a region rich in aromatic amino acids that guide the ligands (ACh or other agonists) by setting an array of low-affinity binding sites. This hydrophobic region traps ACh and transfers it to the deep catalytic site. The catalytic site of AChE consists of two subsites: the "esteratic" site and "the anionic" site. In the "esteratic site" a catalytic triad consisting of E334, H447, S203 forms a planar array that resembles the catalytic triad of serine proteases. S203 is activated (it becomes nucleophilic) by E334 and H447. This activation allows the following reaction: the acylation between hydroxyl group of S203 and ACh oxygen (or other agonists). A covalent bond between the enzyme and the substrate creates an oxyanion. This oxyanion then reacts with two glycins setting up a hydrogen bond. In the "anionic" site, the W86 binds trimethylammonium group of ACh. Further to these steps the substrate is well positioned to be hydrolysed into acetic acid and cholin.
Inhibitors of AChEFasciculin IIFasciculin is a snake toxin. It is a little protein of 7kDa which inhibits AChE in binding the peripheric site, preventing the substrate from passing through the narrower portion of the gorge towards the catalytic site. This inhibition is almost irreversible. The toxin is the one used in cristallisation of the Human acetylcholinesterase (in green on the picture). Inhibitors used as treatmentsWe can find a lot of inhibitors such as in Alzheimer's disease drugs treatment. Actually, Alzheimer's disease is a neurodegenerative disease in which ACh is less present. An inhibition approach can be used to increase the remaining of ACh in the synaptic cleft by inhibiting the action of AChE. These treatments include rivastigmine, donepezil and tacrine. However, these drugs do not cure this disease, but only delay its development. The molecule which has been the most studied is tacrine. A monomer of tacrine binds strongly to the peripheral site, preventing the subtrate from entry. When tacrine is in the dimer shape, it can bind the catalytic and peripheral sites of AChE. Automated computational design of human enzymes for high bacterial expression and stability [2]Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. Using a novel computational strategy, we designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ~2,000-fold higher levels in E. coli compared to wild-type hAChE, and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologues, and is available at https://pross.weizmann.ac.il. The structural underpinnings of stabilization in the designed variant dAChE4. Wild type hAChE is shown in blue and 51 mutated positions, which are distributed throughout dAChE4, are indicated by orange spheres. The choice of mutations at Gly416 in hAChE illustrates the role of these two filters (alignment scan and computational mutation scanning) in pruning false positives (see static image below). Position 416 is located on a partially exposed helical surface, where the small and flexible amino acid Gly is likely to destabilize hAChE. Indeed, in the alignment of 5 AChE homologues, Gly is infrequent and His is the most prevalent amino acid. Modeling shows, however, that in the specific context of hAChE, His adopts a strained side-chain conformation; in contrast, Gln, the third most prevalent amino acid, is predicted to be most stabilizing owing to its high helical propensity and favorable hydrogen-bonding with Tyr504. The combined filter therefore favors Gln over His for downstream design calculations.  Scenes highlight stabilizing effects of selected mutations (in red), wild type hAChE is shown in cyan and designed hAChE is in green: Sub-Ångstrom accuracy in alignment of the catalytic triad Ser203, Glu334, and His447 in the crystallographic structure of dAChE4 (PDB entry: 5hq3, yellow) compared to hAChE (PDB entry: 4ey4, green). Sub-Ångstrom accuracy in alignment of key residues in the vicinity of the catalytic triad.
Comparison of the dAChE4 design model (yellow) with the solved crystal structure (PDB entry: 5hq3, green) and (wild-type hAChE (PDB entry: 4ey4, violet):
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References, for further information on Acetylcholinesterase
To the structures used here:
- Li W, Mak M, Jiang H, Wang Q, Pang Y, Chen K & Han Y (2009) "Novel anti-Alzheimer's dimer Bis(7)-cognitin: cellular and molecular mechanisms of neuroprotection through multiple targets", Neurotherapeutics, vol.6, p.187-201.
- Zhang D & McCammon JA (2005) "The association of tetrameric acetylcholinesterase with colQ tail: a block normal mode analysis", PLoS Comput Biology, vol.1, p.484-491.
To the active site of acetylcholinesterase
- Rosenberry TL (2009) "Strategies to resolve the catalytic mechanism of acetylcholinesterase", Journal of Molecular Neuroscience.
- Currently (November 05, 2009), part of the content of this page is inspired from a source: https://www.biochimie.univ-montp2.fr/licence/enzymo/ache/ache.htm
Hélène ERASIMUS, Blandine FAUVEL, Tiphaine Jaeg 17:08, 12 November 2009 (IST)
