An Unusual Dimeric Inhibitor of Acetylcholinesterase: Cooperative Binding of Crystal Violet

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.

Development of nano Bio LC columns for the search of acetylcholinesterase molecular targets

“…The values of m I, K d , and activity rate (AR) for four other known AChE inhibitors, hup A(‐), hup B(‐), ortho‐7, and apigenin, are given in Table 1. The same order of K d values described in the literature [25–29] was observed: hupA(‐) (0.38 μM) < hupB (0.66 μM) < ortho‐7 (3.64 μM) < hupA(+) (5.75 μM) < apigenin (8.90 μM).…”

Repurposing Triphenylmethane Dyes to Bind to Trimers Derived from Aβ

“…These interactions are similar to the interactions between crystal violet and other proteins observed in high resolution X-ray crystallographic structures. 50,52,53 These structures show that the aryl rings of crystal violet pack against aromatic residues and that the N,N-dimethylamino groups pack against hydrophobic residues. These cocrystal structures do not indicate that the 3-fold symmetry of crystal violet is important for recognition, unlike the trimer 2−crystal violet model.…”

“…Crystal violet is known to interact with numerous proteins, such as BSA and acetylcholine receptors. − ,− For these reasons, we were concerned that the interaction of crystal violet and trimer 2 might not reflect the specific recognition of the 3-fold symmetric dye by the triangular trimer motif. To determine if the association of crystal violet with trimer 2 requires the triangular trimer motif, we studied the interaction of crystal violet with peptide 1 (Figure ).…”

Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study

“…Such structures involving 4-ketoamyltrimethylammonium or choline with AChE [ 4 , 5 ] and acetylcholine with the S203A mutant of AChE [ 5 ] involve simultaneous binding of ligand to the A- and P-sites. Recently, AChE complexes with two or more bound ligands have been deduced from kinetic data for hopeahainol A [ 46 ] and from both kinetic data and the crystal structure with the dye crystal violet [ 47 ]. Neither of these complexes appear to include ligand binding to the A-site, but instead involve simultaneous ligand binding near the entrance of the active site, possibly including the P-site.…”