The interaction of neurotoxin derivatives with either acetylcholine receptor or a monoclonal antibody. An electron-spin-resonance study.
Five derivatives of Naja nigricollis toxin alpha, spin-labeled on a single amino group, were prepared. The toxin derivatives were purified to homogeneity by ion-exchange and high-pressure liquid chromatographies. The modified amino groups are localized at residue 1 and lysines 15, 27, 47 and 51. Competition data show that incorporation of spin label at residues 27 or 47 reduces the affinity of the toxin for the nicotinic acetylcholine receptor (AcChR), while incorporation at residues 1 or 15 diminishes toxin affinity for a monoclonal toxin-specific immunoglobulin (M alpha 1). Classical and/or saturation transfer electron spin resonance (ESR) analysis was carried out on each derivative, either in the free state or bound to AcChR or M alpha 1. The data obtained give the following indications. In the free state, the nitroxides incorporated at residues 1, 15, 47 and 51 have their own rapid motion, while that at residue 27 had no residual mobility and reflects the toxin rotation. Binding of AcChR to the toxin reduces the motion of the nitroxide bound to Lys47. Binding of M alpha 1 to the toxin immobilizes the two nitroxides fixed on residues 1 and 15. ESR spectra show that Lys27-bound nitroxide remains immobilized upon binding of either AcChR or M alpha 1. The change in nitroxide immobilization observed upon AcChR or M alpha 1 binding correlates well with the variation of nitroxide accessibility to a water-soluble paramagnetic N2+i ion. Binding of the labeled Lys47 toxin derivative to AcChR yields a complex ESR signal, disclosing the existence of a physical difference between the two toxin binding sites on AcChR. All the data indicate that AcChR and M alpha 1 bind at two topographically distinct sites on the toxin surface.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000915:Antibody Affinity; D004546:Elapid Venoms / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D009498:Neurotoxins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo
D000911:Antibodies, Monoclonal; D004546:Elapid Venoms; D009498:Neurotoxins; D011950:Receptors, Cholinergic