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.     

Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.