3D Structure of a Complex of Human Acetylcholinesterase with Fasciculin-II at 2.7 Å Resolution

Abstract

A knowledge of the 3D structure of human acetylcholinesterase (AChE) is of importance for the development of anti-Alzheimer drugs, for the general understanding of organophosphate toxicity and for the design of safer and more specific insecticides. A mutant of recombinant human acetylcholinesterase (rhAChE) was constructed in which the C-terminus had been truncated to give rise to a homogeneous monomeric form. The monomeric rhAChE was expressed in HEK 293 cells and purified by affinity chromatog-raphy. The purified enzyme was co-crystallized from ammonium sulfate at pH 7.2 and 19°C as a stoichiometric complex with the mamba venom polypeptide toxin, fasciculin-II (FAS-II). X-ray data were collected at the BNL-NSLS X12-C source from a single cryogenically cooled crystal. The overall completeness of the 30A-2.7Å data is 99.0% and the R-merge is 8.1%. The model was refined starting from the mouse AChE-FAS-II structure to R_free of 29% and R of 21.9% at 2.7Å resolution. The overall fold is similar to that of Torpedo californica and mouse complexes with FAS-II. A mutant E202(199)Q was prepared and crystallized in a similar fashion. Although significantly different from the native enzyme in activity, the E202(199)Q is almost identical to the native in its 3D structure.