Abstract
Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by a rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for treatment of various cholinergic deficiencies, including Alzheimer’s disease and myasthenia gravis. In a previous high throughput screening campaign, we identified the dye crystal violet (CV) as an inhibitor of AChE. Herein, we show that CV displays a significant cooperativity for binding to AChE, and the molecular basis for this observation has been investigated by X-ray crystallography. Two monomers of CV bind to residues at the entrance of the active site gorge of the enzyme. Notably, the two CV molecules have extensive intermolecular contacts with each other and with AChE. Computational analyses show that the observed CV dimer is not stable in solution, suggesting the sequential binding of two monomers. Guided by the structural analysis, we designed a set of single site substitutions, and investigated their effect on the binding of CV. Only moderate effects on the binding and the cooperativity were observed, suggesting a robustness in the interaction between CV and AChE. Taken together, we propose that the dimeric cooperative binding is due to a rare combination of chemical and structural properties of both CV and the AChE molecule itself.
Highlights
The enzyme acetylcholinesterase (AChE) catalyses the hydrolysis of the neurotransmitter acetylcholine, and is an essential component of the central and peripheral nervous system.The pioneering work that led to the first atomic resolution structure of AChE revealed a deep, sterically confined, and highly aromatic active site gorge [1]
Inhibition confirm the ofhigh nH Cooperative observed during the hit confirmation experiments [10], we started by determining of crystal violet (CV) by usingthe wild-type hAChE, Mus musculus[10], AChE
The dose-response curves were analyzed using determining the IC50 values of CV by using wild-type hAChE, Mus musculus AChE, and a Homo four parameters logistic (4PL) model, with a variable slope that allows fittingusing of theaHill sapiens butyrylcholinesterase
Summary
The enzyme acetylcholinesterase (AChE) catalyses the hydrolysis of the neurotransmitter acetylcholine, and is an essential component of the central and peripheral nervous system. The pioneering work that led to the first atomic resolution structure of AChE revealed a deep, sterically confined, and highly aromatic active site gorge [1]. The structure proved a milestone in protein research and has inspired numerous studies of the relation between the structure and dynamics of the active site gorge, and the functional properties of AChE. The molecular recognition of AChE is intricate and typically involves multiple transient binding sites, aromatic interactions, or non-conventional hydrogen bonds [2,3,4,5]. Due to the pivotal role in cholinergic transmission, AChE is an important target for synthetic drugs used for symptomatic treatment of Alzheimer’s disease [6,7], for antidotes used to counteract organophosphorus compounds (i.e., pesticides and nerve agents) [8], and for insecticides used to control vector borne diseases [9].
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