A beta-turn in alpha-amanitin is the most important structural feature for binding to RNA polymerase II and three monoclonal antibodies.
Four amatoxin-binding proteins with KD values in the nanomolar range, 3 monoclonal antibodies and RNA polymerase II, were studied with respect to their affinities to 24 alpha-amanitin derivatives with modified side chains. From KD values we estimated the amounts of binding energy that single side chains of the amatoxins contribute to complex formation. Ile6, previously identified by X-ray analysis to be part of a beta-turn (Kostansek EC, Lipscomb WN, Yocum RR, Thiessen WE, 1978, Biochemistry 17:3790-3795) proved to be of outstanding importance in all complexes. Replacement of the isoleucine with alanine reduced the affinity to all binding proteins to < 1%, suggesting a strong hydrophobic interaction. A strong effect was also seen when Gly5 was replaced with alanine, suggesting that the absence of a side chain in proximity to the beta-turn is likewise important. In addition to the beta-turn, each of the proteins showed at least 2 other points of strong contact formed by hydrogen bonds. Donors are the indole NH of 6'-hydroxy-Trp4 and OH of hydroxy-Pro2 and dihydroxy-Ile3. All the antibodies, but not RNA polymerase II, recognized the indole nucleus of 6'-hydroxy-Trp4. The geometric arrangement of the 4 strongest contact points suggests that the amatoxin binding site is different in each of the 4 proteins, except for the 2 antibodies raised in the same animal. Here, most of the contact points were identical but differed in strength of interaction. The method of structural analysis presented in this study is useful for identifying contact sites in complexes of proteins with peptides of rigid conformation. Furthermore, the method complements X-ray data by providing information on the amount of binding energy contributed by single structural elements.
Baumann
K
K
Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany.
Zanotti
G
G
Faulstich
H
H
D000546:Amanitins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012319:RNA Polymerase II / Q000378:metabolism; D051381:Rats; D013816:Thermodynamics
D000546:Amanitins; D000911:Antibodies, Monoclonal; C018207:amatoxin; D012319:RNA Polymerase II