EXPRESSION AND ACTIVITY OF MUTANTS OF FASCICULIN, A PEPTIDIC ACETYLCHOLINESTERASE INHIBITOR FROM MAMBA VENOM

Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated fro m snake venoms. The availability of a crystal structure of a fasciculi n 2 (Fas2)-acetylcholinesterase complex affords an opportunity to exam ine in detail the interaction of this toxin with its target site. To t his end, we constructed a synthetic fasciculin gene with an appropriat e leader peptide far expression and secretion from mammalian cells. Re combinant wild-type Fas2, expressed and amplified in Chinese hamster o vary cells, was purified to homogeneity and found to be identical in c omposition and biological activities to the venom-derived toxin. Sixte en mutations at positions where the crystal structure of the complex i ndicates a significant interfacial contact point or determinant of con formation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, Delta P ro(30), P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney ce lls. Inhibitory potencies of the Fas2 mutants toward arouse AChE were established, leased on titration of the mutants with a polyclonal anti -Fas2 serum. The Arg(27), Pro(30), and Pro(31) mutants each lost two o r more orders of magnitude in Fas2 activity, suggesting that this subs et of three residues, at the tip of loop II, dominates the loop confor mation and interaction of Fas2 with the enzyme. The Arg(24), Lys(32), and Met(33) mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the com plex, whereas the Lys(25), Arg(28), Val(34)-Leu(35), Asp(45), and Lys( 51) mutants appeared as active as Fas2. The Thr(8)-Thr(9), Arg(11), an d His(29) mutants showed greater ratios of inhibitory activity to immu nochemical titer than Fas2. This may reflect immunodominant determinan ts in these regions or intramolecular rearrangements in conformation t hat enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide sub stantial energetic contributions to the high affinity of the complex.