An .alpha.-Bungarotoxin-Binding Sequence on the Torpedo Nicotinic Acetylcholine Receptor .alpha.-Subunit: Conservative Amino Acid Substitutions Reveal Side-Chain Specific Interactions

Abstract

In the alpha 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 alpha-neurotoxins. Synthetic and biosynthetic peptides corresponding to this region bind alpha-bungarotoxin (alpha-BTX) in the absence of other structural components of the AChR and, therefore, represent a "prototope" for alpha-BTX. Using synthetic peptides corresponding to the complete AChR alpha subunits of Torpedo electroplax and mammalian muscle, we previously defined a sequence segment corresponding to a universal prototope for alpha-BTX binding between amino acid residues 181 and 200 [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R. Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230; McLane, K. E., Wu, X., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544]. To elucidate the structural requirements for alpha-BTX binding, we initially used nonconservative single amino acid substitution analogues of the parental alpha(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 alpha-BTX binding [Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, A., & Maelicke, A. (1991) Biochemistry 30, 2575-2584]. In the present study, amino acid residues previously found to affect alpha-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 alpha-BTX. Whereas V188 could be replaced by Ile or Thr with minor effects on alpha-BTX binding, substitution of Phe, His, or Thr for Y189 and Y190 resulted in large to moderate decreases in alpha-BTX binding. Similarly, alpha-BTX binding activity was intolerant to substitutions of C192 or C193 with Ser, His, or Val. Structural changes of the peptide alpha(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 alpha-BTX binding. Conservative substitutions of other aliphatic and aromatic residues resulted in only minor effects on alpha-BTX binding, as did replacements of K185 and D195 that changed or maintained the charge distribution of peptide alpha (181-200). The recognition site for alpha-BTX formed by the prototope alpha(181-200), therefore, involves important interactions with Y189, Y190, C192, and C193 that are highly specific to the amino acid residue at that position.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of regions involved in the binding of alpha-bungarotoxin to the human alpha7 neuronal nicotinic acetylcholine receptor using synthetic peptides

“…Tyr-188 seems indispensable for α-Bgt binding, as almost no binding to the analogue Y188A was observed ( Figure 3) and a similar role for the homologous Tyr-190 of the Torpedo AChR α1 subunit has been reported [22,40]. Additionally, the homologous Tyr of the chick α7 αChR (Tyr-187) is crucial for the binding of α-cobratoxin and α-Bgt.…”

“…Three of these corresponded to the three main loops of the toxin, i.e. peptide α-Bgt (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) corresponded to loop I, α-Bgt (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41) to part of loop II, and α-Bgt (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59) to loop III, while the other two included the C-terminal tail of the toxin (residues 68-74) plus part of the core [peptide α-Bgt(58-74)], or another part of the core plus one side of loop II [peptide α-Bgt …”

“…Results showed that toxin binding to α7 AChR and inhibition percentages with peptides were not affected by the use of BSA. Similarly, when these soluble α-Bgt peptides were tested for their ability to inhibit 125 I-α-Bgt binding to membranebound Torpedo AChR, they inhibited 125 I-α-Bgt binding in a concentration-dependent manner, 50 % inhibition being achieved with 0.07 mg/ml α-Bgt(58-74) and with 0.22 mg/ml α-Bgt (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41) (Figure 7). These peptides were also tested using solubilized Torpedo AChR but no significant differences from results with the membrane-bound AChR were evident (results not shown).…”

“…Overall, the results demonstrated that loop II (peptide [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] and the C-terminal tail (peptide 58-74) of α-Bgt are the major regions by which the toxin makes contact with both α7 and muscle-type receptor ( Figure 6). No binding to loop I or III was observed, indicating the absence of significant binding determinants at these regions.…”

Novelty in three-finger toxin evolution

“…As per previous studies [231], the cysteine doublet in the orthosteric binding site sequence was replaced in peptide synthesis steps with serine doublet to avoid uncontrolled postsynthetic thiol oxidation. Research has shown this has no effect on the analyte-ligand complex formation [250][251][252]. The mimotope was further synthesised to a biotin linker bound to a two aminohexanoic acid (Ahx) spacers to form a 30Å linker between biotin and the peptide.…”