An a-bungarotoxin-binding sequence on the Torpedo nicotinic acetylcholine receptor a-subunit: conservative amino acid substitutions reveal side-chain specific interactions

Mclane, K.E.; Wu, X.; Conti Tronconi, B.M.

Biochemistry (American Chemical Society) 33: 76-85

1994


Accession: 009766016

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Abstract
In the a subunit of the Torpedo nicotinic cholinergic receptor (AChR), a sequence region surrounding a pair of adjacent cysteinyl residues at positions 192 and 193 contributes to a binding site for cholinergic ligands, including the snake a-neurotoxins. Synthetic and biosynthetic peptides corresponding to this region bind a-bungarotoxin (a-BTX) in the absence of other structural components of the AChR and, therefore, represent a "prototope" for a-BTX. Using synthetic peptides corresponding to the complete AChR a subunits of Torpedo electroplax and mammalian muscle, we previously defined a sequence segment corresponding to a universal prototope for a-BTX binding between amino acid residues 181 and 200 [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R. Reinhardt-Maelicke, S., and Maelicke, A. (1990) Biochemistry 29, 6221-6230; McLane, K. E., Wu, X., and Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544]. To elucidate the structural requirements for a-BTX binding, we initially used nonconservative single amino acid substitution analogues of the parental a(181-200) sequence, and we found that residues at positions 188-190 (VYY), and 192-194 (CCP) and several flanking residues seemed to be involved in a-BTX binding [Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, A., and Maelicke, A. (1991) Biochemistry 30, 2575-2584]. In the present study, amino acid residues previously found to affect a-BTX binding were replaced by different conservative single amino acid substitutions, in order to determine the nature of the amino acid side-chain interactions with a-BTX. Whereas V188 could be replaced by Ile or Thr with minor effects on a-BTX binding, substitution of Phe, His, or Thr for Y189 and Y190 resulted in large to moderate decreases in a-BTX binding. Similarly, a-BTX binding activity was intolerant to substitutions of C192 or C193 with Ser, His, or Val. Structural changes of the peptide a(181-200) induced by substitution of P194 or P197 with two adjacent Gly residues, and insertion of a Gly between C192 and C193, were also incompatible with a-BTX binding. Conservative substitutions of other aliphatic and aromatic residues resulted in only minor effects on a-BTX binding, as did replacements of K 185 and D 195 that changed or maintained the charge distribution of peptide a(181-200). The recognition site for a-BTX formed by the prototope a(181-200), therefore, involves important interactions with Y189, Y190, C192, and C193 that are highly specific to the amino acid residue at that position. Furthermore, P194 and P197 appear to play important structural roles in maintaining the correct conformation of the peptide to display this binding motif. Copyright 1994, American Chemical Society. .