STRUCTURE-FUNCTION-RELATIONSHIPS OF CURAREMIMETIC NEUROTOXIN LOOP-2 AND OF A STRUCTURALLY SIMILAR SEGMENT OF RABIES VIRUS GLYCOPROTEIN IN THEIR INTERACTION WITH THE NICOTINIC ACETYLCHOLINE-RECEPTOR

Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of I-125-alpha-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b (IC50 = 5.7 x 10(-6) M) and the structurally similar segment (residues 173-203) of CVS rabies virus glycoprotein (IC50 = 2.6 x 10(-6) M). These affinities were comparable to those of d-tubocurarine (IC50 = 3.4 x 10(-6) M) and suberyldicholine (IC50 = 2.5 X 10(-6) M). These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Modifications involving Lys-27, Trp-29, Phe-33, Arg-37, and Gly-38 reduced affinity of binding. R37D and F33T modifications reduced the affinity of alpha-bungarotoxin residues 28-40 by an order of magnitude. Arg-37 may correspond to the positively charged quaternary ammonium group and Phe-33 to the hydrophobic acetyl methyl group of acetylcholine. These residues may interact with complementary electronegative and hydrophobic sites, respectively, on the receptor alpha-subunit surface in the vicinity of Cys-192 and Cys-193. The largest decrease in affinity (approximately 20-fold) followed substitution of Gly-38 with a tryptophan residue. The introduction of a bulky indole side chain at this site may sterically interfere with the interaction of Arg-37 with an anionic site on the receptor. Substitution of Asp-31, a potential counterpart of the hydrogen bond acceptor formed by the carbonyl oxygen of acetylcholine, with an alanine had no effect on affinity. The rabies virus glycoprotein synthetic peptides acted in a manner very similar to that of the neurotoxin peptides. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.