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 125I-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Lentz
T L
TL
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D004546:Elapid Venoms / Q000378:metabolism* / Q000494:pharmacology; D004557:Electric Organ / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000378:metabolism* / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D011820:Rabies virus / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014101:Torpedo; D014403:Tubocurarine / Q000494:pharmacology*; D014764:Viral Proteins / Q000378:metabolism* / Q000494:pharmacology
D004546:Elapid Venoms; D006023:Glycoproteins; D046911:Macromolecular Substances; D009498:Neurotoxins; D010455:Peptides; D011978:Receptors, Nicotinic; D014764:Viral Proteins; D014403:Tubocurarine