Variability among the sites by which curaremimetic toxins bind to Torpedo acetylcholine receptor, as revealed by identification of the functional residues of alpha-cobratoxin

alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom , was produced recombinantly (r alpha-Cbtx) from Escherichia coli. It was i ndistinguishable from the snake toxin, Mutations at 8 of the 29 explored to xin positions resulted in affinity decreases for Torpedo receptor with Delt a Delta G higher than 1.1 kcal/mol, These are R33E > K49E > D27R > K23E > F 29A greater than or equal to W25A > R36A greater than or equal to F65A. The se positions cover a homogeneous surface of approximately 880 Angstrom(2) a nd mostly belong to the second toxin loop, except Lys-49 and Phe-65 which a re, respectively, on the third loop and C-terminal tail. The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites. When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in bi nding to muscular-type nicotinic acetylcholine receptor. These are Lys-23/L ys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and var iable extent Trp-25/Trp-29 and Phe-29/Phe-32. In addition, however, the sho rt and long toxins display three major differences. First, Asp-38 is import ant in Ea in contrast to the homologous Glu-38 in alpha-Cbtx, Second, all o f the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea. Third, the C-terminal tail may be specifica lly critical in alpha-Cbtx. Therefore, the functional sites of long and sho rt curaremimetic toxins are not identical, but they share common features a nd marked differences that might reflect an evolutionary pressure associate d with a great diversity of prey receptors.