The Mechanism for Acetylcholine Receptor Inhibition by α-Neurotoxins and Species-Specific Resistance to α-Bungarotoxin Revealed by NMR

or ␦ (i.e ␣1 X), respectively, before the one letter amino acid code indicating the subunit type and the position in sequence). Sequence analysis has shown that resistance of certain species such as mongoose and cobra to ␣-BTX is correlated with mutations in this region (Bar-In comparison to Torpedo AChR, which is homolo-Rehovot 76100 Israel gous to muscle AChR, the neuronal AChR (␣7-homopen-tamer) binds ␣-BTX with 20-fold weaker affinity and short neurotoxins with five orders of magnitude weaker affinity (Servent et al., 1998). ␣7 does not show any Summary significant variations in its amino acid sequence among different species and even mongoose ␣7 binds ␣-BTX The structure of a peptide corresponding to residues (Ariel et al., 1998). The peptide ␣7 181-200 of chick neuronal 182–202 of the acetylcholine receptor ␣1 subunit in AChR (IPGKRNESFYECCKEPYPD, the numbering of all complex with ␣-bungarotoxin was solved using NMR peptides is according to alignment with ␣1) binds ␣-BTX spectroscopy. The peptide contains the complete se-with a dissociation constant of 3 ϫ 10 Ϫ5 M, three orders quence of the major determinant of AChR involved in of magnitude higher than that for the ␣1 187-200 peptide ␣-bungarotoxin binding. One face of the long ␤ hairpin (Harel et al., 2001; Moise et al., 2002). formed by the AChR peptide consists of exposed non-The three-dimensional solution structure of ␣-BTX in conserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer complex with a 13 residue peptide, LP (MRYYESSLK resistance to the toxin. Conserved AChR residues SYPD), selected from a phage-displayed random pep-form the opposite face of the ␤ hairpin, which creates tide library (Scherf et al., 1997) as well as that between the inner and partially hidden pocket for acetylcholine. ␣-BTX and two peptides based on the library peptide An NMR-derived model for the receptor complex with and modified for higher affinity, HAP2 and HAP (WRYY two ␣-bungarotoxin molecules shows that this pocket ESSLEPYPD and WRYYESSLLPYPD, respectively), is occupied by the conserved ␣-neurotoxin residue were determined by NMR (HAP2) and X-ray crystallogra-R36, which forms cation-␲ interactions with both phy (HAP) (Harel et al., 2001; Scherf et al., 2001). In the ␣ W149 and ␥ W55/ ␦ W57 of the receptor and mimics ace-latter two structures, nine peptide residues were found tylcholine. to interact with ␣-BTX. The high-affinity peptides are highly homologous to the major ␣-BTX binding determi-Introduction nant of the ␣7 subunit (␣7 …