INHIBITION OF MOUSE ACETYLCHOLINESTERASE BY FASCICULIN - CRYSTAL-STRUCTURE OF THE COMPLEX AND MUTAGENESIS OF FASCICULIN

Fasciculins are members of the superfamily of three-fingered peptidic toxins from Elapidae venoms. They selectively inhibit mammalian and el ectric fish acetylcholinesterases (AChE) with K-i values in the pico- to nanomolar range. Kinetic studies performed in solution indicate tha t fasciculin does not totally occlude ligand access to the active site of AChE, but rather binds to a peripheral site of the enzyme to inhib it catalysis, perhaps allosterically. The crystal structure of the Fas 2-mouse AChE complex delineated a large contact area consistent with t he low dissociation constant of the complex; the Fas2 and AChE residue s participating in the binding interface were unambiguously establishe d, and major hydrophobic interactions were identified. The structure h owever suggests that fasciculin totally occludes substrate entry into the catalytic site of AChE, and does not reveal to what extent each co ntact between Fas2 and AChE contributes to the overall binding energy. New probes, designed to delineate the individual contributions of the fasciculin residues to the complex formation and conformation, were g enerated by site-directed mutagenesis of a synthetic Fas2 gene. A full y processed recombinant fasciculin, rFas2, that is undistinguishable f rom the natural, venom-derived Fas2, was expressed in a mammalian syst em; fourteen mutants, encompassing 16 amino acid residues distributed among the three loops (fingers) of Fas2, were developed from both the kinetic and structural data and analyzed for inhibition of mouse AChE. The determinants identified by the structural and the functional appr oaches do coincide. However, only a few of the many residues which mak e up the overall interactive site of the Fas2 molecule provide the str ong interactions required for high affinity binding and enzyme inhibit ion. Potential drug design from the fasciculin molecule is discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.