Interaction of protein ligands with receptor fragments. On the residues of curaremimetic toxins that recognize fragments 128-142 and 185-199 of the alpha-subunit of the nicotinic acetylcholine receptor.
Using a solid-phase assay, we found that 3H-labeled alpha Cobtx from Naja naja siamensis, a long-chain curaremimetic toxin, and 3H-labelled toxin alpha from Naja nigricollis, a short-chain toxin both bind specifically but with substantially different affinities (Kd = 4 x 10(-7) M and 50 x 10(-6) M) to fragment 185-199 (T alpha 185-199) of the alpha-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 T alpha 185-199, suggesting that none of these invariant residues in implicated in the recognition of this AcChoR region. alpha Cobtx and toxin alpha bind to the fragment 128-142 (T alpha 128-142) with more similar affinities (Kd = 3 x 10(-7) M and 1.4 x 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.
Fulachier
M H
MH
Département d'Ingénierie et d'Etudes des Protéines, C.E.A., Saclay, Gif/Yvette, France.
Mourier
G
G
Cotton
J
J
Servent
D
D
Ménez
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D003473:Neuromuscular Nondepolarizing Agents / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo
D003039:Cobra Neurotoxin Proteins; D008024:Ligands; D003473:Neuromuscular Nondepolarizing Agents; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; C025154:alpha-cobratoxin