The alpha-bungarotoxin binding site on the nicotinic acetylcholine receptor: analysis using a phage-epitope library.
The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. alpha-Neurotoxins, in particular alpha-bungarotoxin (alpha-BTX), bind specifically and with high affinity to the AcChoR and compete with binding of the natural ligand. We employed a 15-mer phage-display peptide library to select epitopes reacting with alpha-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to alpha-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the alpha-subunit of the Torpedo muscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind alpha-BTX. The library-derived peptide and the corresponding peptides containing residues 187-199 of the Torpedo AcChoR alpha-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human alpha7; ERFYECCKEPYPD) that binds alpha-BTX, inhibit the binding of alpha-BTX to the intact Torpedo AcChoR with IC50 values of 10(-6) M. A synthetic peptide from a neuronal AcChoR that does not bind alpha-BTX (e.g., human alpha2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the alpha-BTX-binding protein (alpha7)-i.e., Lys in alpha2 and Tyr in alpha7-does not inhibit the binding of alpha-BTX to Torpedo AcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to alpha-BTX.
Balass
M
M
Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel.
Katchalski-Katzir
E
E
Fuchs
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism*; D000939:Epitopes; D006801:Humans; D007256:Information Systems; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014101:Torpedo
D002038:Bungarotoxins; D000939:Epitopes; D046911:Macromolecular Substances; D009842:Oligopeptides; D011978:Receptors, Nicotinic