Identification of structural features involved in binding of alpha-amanitin to a monoclonal antibody.
Twenty-four derivatives of the cyclic octapeptide alpha-amanitin were assayed for their affinities 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 microM. 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 hydroxyproline2 (binding factor 413), (2) the lipophilic side chain of isoleucine6 (binding factor 131), (3) the -CH2- moiety of the adjacent glycine5 or the absence of a side chain in this position (binding factor 361), and (4) the proton at the indole nitrogen of hydroxytryptophan4 (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.
Baumann
K
K
Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany.
Münter
K
K
Faulstich
H
H
D000546:Amanitins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D002942:Circular Dichroism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008745:Methylation; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar / Q000276:immunology; D013329:Structure-Activity Relationship
D000546:Amanitins; D000911:Antibodies, Monoclonal