NMR-based binding screen and structural analysis of the complex formed between α-cobratoxin and an 18-mer cognate peptide derived from the α1 subunit of the nicotinic acetylcholine receptor from torpedo californica (original) (raw)

2002, Journal of Biological Chemistry

The ␣18-mer peptide, spanning residues 181-198 of the Torpedo nicotinic acetylcholine receptor ␣1 subunit, contains key binding determinants for agonists and competitive antagonists. To investigate whether the ␣18-mer can bind other ␣-neurotoxins besides ␣-bungarotoxin, we designed a two-dimensional 1 H-15 N heteronuclear single quantum correlation experiment to screen four related neurotoxins for their binding ability to the peptide. Of the four toxins tested (erabutoxin a, erabutoxin b, LSIII, and ␣-cobratoxin), only ␣-cobratoxin binds the ␣18-mer to form a 1:1 complex. The NMR solution structure of the ␣-cobratoxin⅐␣18-mer complex was determined with a backbone root mean square deviation of 1.46 Å. In the structure, ␣-cobratoxin contacts the ␣18-mer at the tips of loop I and II and through C-terminal cationic residues. The contact zone derived from the intermolecular nuclear Overhauser effects is in agreement with recent biochemical data. Furthermore, the structural models support the involvement of cation-interactions in stabilizing the complex. In addition, the binding screen results suggest that C-terminal cationic residues of ␣-bungarotoxin and ␣-cobratoxin contribute significantly to binding of the ␣18-mer. Finally, we present a structural model for nicotinic acetylcholine receptor-␣-cobratoxin interaction by superimposing the ␣-cobratoxin⅐␣18-mer complex onto the crystal structure of the acetylcholine-binding protein (Protein Data Bank code 1I9B).