The Complex of a Bivalent Derivative of Galanthamine with Torpedo Acetylcholinesterase Displays Drastic Deformation of the Active-Site Gorge:  Implications for Structure-Based Drug Design

Abstract

Bifunctional derivatives of the alkaloid galanthamine, designed to interact with both the active site of the enzyme acetylcholinesterase (AChE) and its peripheral cation binding site, have been assayed with Torpedo californica AChE (TcAChE), and the three-dimensional structures of their complexes with the enzyme have been solved by X-ray crystallography. Differences were noted between the IC(50) values obtained for TcAChE and those for Electrophorus electricus AChE. These differences are ascribed to sequence differences in one or two residues lining the active-site gorge of the enzyme. The binding of one of the inhibitors disrupts the native conformation of one wall of the gorge, formed by the loop Trp279-Phe290. It is proposed that flexibility of this loop may permit the binding of inhibitors such as galanthamine, which are too bulky to penetrate the narrow neck of the gorge formed by Tyr121 and Phe330 as seen in the crystal structure.

Monitoring enzyme reaction and screening of inhibitors of acetylcholinesterase by quantitative matrix-assisted laser desorption/ionization fourier transform mass spectrometry

“…The inhibition plot of galantamine is shown in Figure 5b. The IC 50 value of galantamine was determined as 2.39 M, which was comparable with the literature values of 0.42 to 5 M (0.42 M for Torpedo californica AChE [44], 0.61 M for Electrophorus electricus AChE [44], 5 M for AChE in postmortem brain tissue from AD patients [45]). …”

Crystal structures of oxime-bound fenamiphos-acetylcholinesterases: Reactivation involving flipping of the His447 ring to form a reactive Glu334–His447–oxime triad

“…An adaptable central linker is consistent with the previous suggestion that Ortho-7 can adjust to different conformations of the active-site gorge [7]. Moreover, the acyl loop has previously been described as flexible and various reversible and irreversible ligands are known to induce a structural change in this region [29,[37][38][39]. For example, side-chain rotations of Phe295 and Phe297, similar to the movement observed in Ortho-7fep-mAChE, have been described in the bis-tacrine-bound Torpedo californica AChE complex (pdb code: 2cmf; [39]).…”

Shoot-and-Trap: Use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography

“…The native structure of TcAChE in the orthorhombic space group (PDB ID code 1W75) (41) served as the starting model for the rigid body refinement of data set I 100K in the resolution range 50 -4 Å. Subsequently, the structure underwent simulated annealing to 2,000 K, with slow-cooling steps of 10 K, followed by 250 steps of conjugategradient minimization and 20 steps of individual B-factor refinement.…”