.alpha.-Bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite

Abstract

In the nicotinic acetylcholine receptors (AChRs), the sequence segment surrounding two invariant vicinal cysteinyl residues at positions 192 and 193 of the alpha subunit contains important structural component(s) of the binding site for acetylcholine and high molecular weight cholinergic antagonists, like snake alpha-neurotoxins. At least a second sequence region contributes to the formation of the cholinergic site. Studying the binding of alpha-bungarotoxin and three different monoclonal antibodies, able to compete with alpha-neurotoxins and cholinergic ligands, to a panel of synthetic peptides as representative structural elements of the AChR from Torpedo, we recently identified the sequence segments alpha 181-200 and alpha 55-74 as contributing to form the cholinergic site (Conti-Tronconi et al., 1990). As a first attempt to elucidate the structural requirements for ligand binding to the subsite formed by the sequence alpha 181-200, we have now studied the binding of alpha-bungarotoxin and of antibody WF6 to the synthetic peptide alpha 181-200, and to a panel of peptide analogues differing from the parental sequence alpha 181-200 by substitution of a single amino acid residue. CD spectral analysis of the synthetic peptide analogues indicated that they all have comparable structures in solution, and they can therefore be used to analyze the influence of single amino acid residues on ligand binding. Distinct clusters of amino acid residues, discontinuously positioned along the sequence 181-200, seem to serve as attachment points for the two ligands studied, and the residues necessary for binding of alpha-bungarotoxin are different from those crucial for binding of antibody WF6. In particular, residues at positions 188-190 (VYY) and 192-194 (CCP) were necessary for binding of alpha-bungarotoxin, while residues W187, T191, and Y198 and the three residues at positions 193-195 (CPD) were necessary for binding of WF6. Comparison of the CD spectra of the toxin/peptide complexes, and those obtained for the same peptides and alpha-bungarotoxin in solution, indicates that structural changes of the ligand(s) occur upon binding, with a net increase of the beta-structure component. The cholinergic binding site is therefore a complex surface area, formed by discontinuous clusters of amino acid residues from different sequence regions. Such complex structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody/antigen complexes [reviewed in Davies et al. (1988)]. Within this relatively large structure, cholinergic ligands bind with multiple points of attachment, and ligand-specific patterns of the attachment points exist.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of protein ligands with receptor fragments

“…That the fragment Ta185-199 1s implicated m binding of long-chain curaremlmetlc toxins 1s m agreement with the literature [20,21,23,24,26,27,34,36] We now show that a short-chain toxin also recognizes this region but with lower affinity This finding contrasts with data from Ruan et al [33] who reported that 'region a182-198 was not a bmdmg region for short-neurotoxins' However, these authors used a different bmdmg protocol and the peptlde used m their study had no dlsulfide, a factor that may greatly influence the toxin bmdmg…”

“…More precise defimtion of the curaremimetic toxin bmdmg site has been attempted wtth fragments of a subunits generated as proteolytic [15-l 81, synthetic [19][20][21][22][23][24][25][26][27] or fusion peptides obtained by genetic means It is accepted that the region around the cysteme 192-193 is recognized by most AcChoR hgands. Systematic replacement of the residues by alanme suggest a special contribution of residues Tyr-190, Cys-192, Cys-193, Val-188, Thr-189, to formatton of toxin-peptide complexes [31].…”

Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor α Subunit in Yeast Pichia pastoris

“…(iii) ␣-BTX bound to h␣-(1-210) with a high affinity (K d ϭ 5.1 Ϯ 2.4 nM). High affinity binding of ␣-BTX to the AChR ␣ subunit is routinely used to indicate correct folding of the recombinant molecule as the formation of the binding site requires the coordination of several post-translational events that draw together different regions of the ␣ subunit (11,12). (iv) The small competitive antagonists, dtubocurarine and gallamine, inhibited ␣-BTX binding to h␣- .…”

“…Previous experiments have shown that the binding sites for both acetylcholine and ␣-BTX are located close to two adjacent cysteine residues at positions 192 and 193 of the ␣ subunit (10). Moreover, other distinct regions on either the ␣ subunit (11,12) or the adjacent ␥ or ␦ subunits (13) have been shown to contribute to ␣-BTX binding, whereas the role of glycosylation at residue ␣141 (14 -16) requires further study.…”

Application of an ectopic expression system for the selection of protein‐isoform‐specific antibodies

“…Initial screening of hybridoma cells was by ELISA [15], emplclying fusion protein I1 (0.05 pg/well of the microtiter plate)…”