Structural determinants of .alpha.-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor .alpha. subunit: effects of cysteine/cystine modification and species-specific amino acid substitutions

Abstract

The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha subunit forms a binding site for alpha-bungarotoxin (alpha-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR alpha 1 subunits were tested for their ability to bind 125I-alpha-BTX, and differences in alpha-BTX affinity were determined by using solution (IC50S) and solid-phase (KdS) assays. Panels of overlapping peptides corresponding to the complete alpha 1 subunit of mouse and human were also tested for alpha-BTX binding, but other sequence segments forming the alpha-BTX site were not consistently detectable. The Torpedo alpha 1(181-200) and the homologous frog and chicken peptides bound alpha-BTX with higher affinity (KdS approximately 1-2 microM, IC50s approximately 1-2 microM) than the human and calf peptides (Kds approximately 3-5 microM, IC50s approximately 15 microM). The mouse peptide bound alpha-BTX weakly when attached to a solid support (Kd approximately 8 microM) but was effective in competing for 125I-alpha-BTX in solution (IC50 approximately 1 microM). The cobra nAChR alpha 1-subunit peptide did not detectably bind alpha-BTX in either assay. Amino acid substitutions were correlated with alpha-BTX binding activity peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased alpha-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce alpha-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/alpha 1-subunit interface a vicinal disulfide bound was not required for alpha-BTX binding.

Structural determinants within residues 180-199 of the rodent .alpha.5 nicotinic acetylcholine receptor subunit involved in .alpha.-bungarotoxin binding

“…A disulfide bridge between flanking cysteine residues requires a cis, nonplanar peptide bond, which is an unusual configuration likely to alter the reactivity of the sensitive cystine (Dayhoff, 1976;Schultz & Schirmer, 1979;Thorton, 1981;Ovchinnikov et al, 1985Ovchinnikov et al, , 1988. On the other hand, we found here, in agreement with previous determinations of the homologous Torpedo and vertebrate muscle peptides (Conti-Tronconi et al, 1991;McLane et al, 1991a), that -30% of the Cys residues of the untreated peptide a5 ( 180-199),,a,ive are oxidized as disulfide bridges and as the amount of dimer present in these preparations is insufficient to account for the total peptide present in oxidized form we must conclude that a disulfide bridge forms spontaneously between the vicinal Cys of the peptide. The role of vicinal disulfide in a-BTX binding to the Torpedo a1 sequence, however, remains equivocal.…”

“…In addition, the cobra muscle nAChR is unique among the vertebrate muscle nAChRs in that it is insensitive to a-BTX (Burden et al, 1975), and synthetic peptides corresponding to the sequence segment 181-200 of the a1 subunit of different species have been shown to bind a-BTX using the Torpedo competition assay, with the exception of the cobra sequence, which differs by only a few amino acid residues (McLane et al, 1991a).…”

An Appetite for Destruction: Detecting Prey-Selective Binding of α-Neurotoxins in the Venom of Afro-Asian Elapids

“…The Cys-Cys of the native mimotope was replaced during peptide synthesis with Ser-Ser to avoid uncontrolled postsynthetic thiol oxidation. The Cys-Cys bond in the nAChR binding region does not participate directly in analyte-ligand binding [57][58][59], thus replacement to Ser-Ser is not expected to have any effect on the analyte-ligand complex formation. However, the presence of the Cys-Cys bridge is key in the conformation of the interaction site of whole receptors [60].…”

Assessing the Binding of Venoms from Aquatic Elapids to the Nicotinic Acetylcholine Receptor Orthosteric Site of Different Prey Models

“…The Cys–Cys of the native mimotope was replaced during peptide synthesis with Ser-Ser to avoid uncontrolled postsynthetic thiol oxidation. The Cys–Cys bond in the nAChR binding region does not participate directly in analyte–ligand binding [ 20 , 73 , 74 ], thus, replacement to Ser-Ser is not expected to have any effect on the analyte–ligand complex formation. However, the presence of the Cys–Cys bridge is key in the conformation of the interaction site of whole receptors [ 75 ].…”