Interaction of protein ligands with receptor fragments

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Abstract
Using a solid‐phase assay, we found that 3H‐labelled αCobtx from Naja naja siamensis, a long‐chain curaremimetic toxin, and 3H‐labelled toxin a from Naja nigricollis, a short‐chain toxin both bind specifically but with substantially different affinities (K d = 4·10−7 M and 50·10−6M) to fragment 185–199 (Tα185–199) of the α‐subunit of the acetylcholine receptor (AcChoR) from Torpedo marmorata. Then we show that monoderivatizations of residues common to both long‐chain and short‐chain toxins (Tyr‐25, Lys‐27, Trp‐29, and Lys‐53) or to long‐chain toxins only (Cys‐30 and Cys‐34) do not affect the binding of the toxins to Tα185‐199, suggesting that none of these invariant residues is implicated in the recognition of this AcChoR region. αCobtx and toxin α bind to the fragment 128‐142 (Tα128‐142) with more similar affinities (K d = 3·10−7 M and 1.4·10−6 M) and their binding is dramatically affected by the single abolition of the positive charge of Lys‐53, an invariant residue that contributes to AcChoR recognition. Therefore, the data indicate that Lys‐53 more specifically recognizes the 128–142 region of AcChoR. Other monoderivatizations have no effect on toxin binding. The approach described in this paper may be of great help to identify toxin residues that establish direct contact with receptor fragments.

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