Compatibility of Structures Inferred from Mutagenesis and from X-Ray Crystallography for Various AChE Complexes

Abstract

Interactions of the potential therapeutic agents tacrine, huperzine A and edrophonium, with 15 mutants of human acetylcholinesterase (HuAChE) were investigated. The score of new inhibition and dissociation constants measured, expand considerably the corresponding data found in literature. Despite the structural diversity of the ligands certain common properties of the complexes could be observed: a. replacement of aromatic residues Y133, Y337 and especially W86, resulted in pronounced changes in stability of all the complexes examined, b. effects due to replacements of the five other aromatic residues along the active-center gorge such as the acyl pocket (F295, F297) or at the peripheral anionic site (Y124, W286, Y341) were relatively small, c. effects due to substitution of the carboxylic residues in the gorge (E202, E450) were moderate. These results and the corresponding molecular models indicate that the aromatic side chains of residues W86, Y133 and Y337 form together a continuous “aromatic patch” lining the wall of the active center gorge allowing for the accommodation of the different ligands via various modes of interactions (cation-π, π-π, H-bond, hydrophobic). The HuAChE-bound orientations of edrophonium, huperzine A and tacrine inferred from the mutagenesis studies, were compared to those in the available X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). For edrophonium the two structures are very similar with respect to the ligand and its binding environment. On the other hand, the x-ray orientation for tacrine and huperzine A is inconsistent with mutagenesis results - some mutations do not support expected interactions while others suggest interactions that are not observed in the x-ray structures. These inconsistencies may be explained in terms of: a. structural differences between the complexes of HuAChE and TcAChE, even though the active center regions of the two enzymes, consisting of over 30 residues, differ in only one position [Y337(F330)]; b. the active-center gorge of AChE in solution is more flexible than in the crystalline state allowing for modified binding orientations of the same ligand in the two states.