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 fromTorpedo californica
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,
... 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).