AN ALPHA-BUNGAROTOXIN-BINDING SEQUENCE ON THE TORPEDO NICOTINIC ACETYLCHOLINE-RECEPTOR ALPHA-SUBUNIT - CONSERVATIVE AMINO-ACID SUBSTITUTIONS REVEAL SIDE-CHAIN SPECIFIC INTERACTIONS

In the alpha subunit of the Torpedo nicotinic cholinergic receptor (AC hR), a sequence region surrounding a pair of adjacent cysteinyl residu es at positions 192 and 193 contributes to a binding site for choliner gic ligands, including the snake alpha-neurotoxins. Synthetic and bios ynthetic 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 synthe tic peptides corresponding to the complete AChR alpha subunits of Torp edo 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 [ContiTronconi, 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 th e structural requirements for alpha-BTX binding, we initially used non conservative single amino acid substitution analogues of the parental alpha(181-200) sequence, and we found that residues at positions 188-1 90 (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 3 0, 2575-2584]. In the present study, amino acid residues previously fo und to affect alpha-BTX binding were replaced by different conservativ e single amino acid substitutions, in order to determine the nature of the amino acid side-chain interactions with alpha-BTX. Whereas V188 c ould 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 bind ing 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 i nsertion of a Gly between C192 and C193, were also incompatible with a lpha-BTX binding. Conservative substitutions of other aliphatic and ar omatic residues resulted in only minor effects on alpha-BTX binding, a s did replacements of K185 and D195 that changed or maintained the cha rge distribution of peptide alpha(181-200). The recognition site for a lpha-BTX formed by the prototope alpha(181-200), therefore, involves i mportant 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.