Identification of structural features involved in binding of .alpha.-amanitin to a monoclonal antibody

• Published in: Biochemistry (Easton) Vol. 32; no. 15; pp. 4043 - 4050
• Main Authors: Baumann, K, Muenter, K, Faulstich, H
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 20.04.1993
• Subjects: AMANITA; Amanitins - chemistry; Amanitins - immunology; Amanitins - metabolism; Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Animals; Antibodies, Monoclonal - isolation & purification; Antibodies, Monoclonal - metabolism; ANTICORPS MONOCLONAL; ANTICUERPOS MONOCLONALES; Binding, Competitive; Biological and medical sciences; Circular Dichroism; COMPOSE AMINE; COMPUESTOS DE AMINA; Fundamental and applied biological sciences. Psychology; Kinetics; Magnetic Resonance Spectroscopy - methods; Methylation; MICOTOXINAS; MYCOTOXINE; Protein Conformation; Proteins; Rats; Rats, Wistar - immunology; REACCIONES QUIMICAS; REACTION CHIMIQUE; Structure-Activity Relationship; Molecular interaction; Binding site; Monoclonal antibody; Cyclic peptides
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00066a027

Twenty-four derivatives of the cyclic octapeptide alpha-amanitin were as to the monoclonal antibody beta-A1/1. The derivatives were of natural, semisynthetic, and synthetic origin and had KD values ranging from 2 nM to 70 micromolars. In the majority of the derivatives the chemical modifications had no detectable influence on the overall shape of the double-ring peptide. Given this condition, binding factors could be calculated from KD values of the amatoxin derivatives, which were valid for all amatoxins for estimating the contribution made by single structures to complex formation. The complex between alpha-amanitin and the immunoglobulin involves at least eight sites of contact. Four of them are responsible for strong interactions: (1) the OH group of hydroxyproline (binding factor 413), (2) the lipophilic side chain of isoleucine (binding factor 131), (3) the -CH2--moiety of the adjacent glycine or the absence of a side chain in this position (binding factor 361), and (4) the proton at the indole nitrogen of hydroxytryptophan (binding factor 140). The residual four interactions are hydrogen bonds of lower strength corresponding to binding factors of 1.5-8. The key role of the unique conformation of the amatoxins in determining their binding properties was shown by two amatoxin derivatives in which changes in the conformation were associated with virtually complete loss of affinity. For all amatoxin derivatives with conformations similar or identical to that of alpha-amanitin, we found empirical evidence that those structures of the peptide involved in binding make their contributions virtually independent of each other. It is a consequence of this rule that structural features that cooperate in binding could be characterized by the numerical product of their binding factors