title: 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)
authors.affiliation: Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul 55108.
authors.forename: B M
authors.initials: BM
authors.lastname: Conti-Tronconi
authors.forename: B M
authors.initials: BM
authors.lastname: Diethelm
authors.forename: X D
authors.initials: XD
authors.lastname: Wu
authors.forename: F
authors.initials: F
authors.lastname: Tang
authors.forename: T
authors.initials: T
authors.lastname: Bertazzon
authors.forename: B
authors.initials: B
authors.lastname: Schröder
authors.forename: S
authors.initials: S
authors.lastname: Reinhardt-Maelicke
authors.forename: A
authors.initials: A
authors.lastname: Maelicke
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D002038:Bungarotoxins / Q000276:immunology / Q000378:metabolism*; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic