ENGINEERING OF PROTEIN EPITOPES - A SINGLE DELETION IN A SNAKE TOXIN GENERATES FULL BINDING-CAPACITY TO A PREVIOUSLY UNRECOGNIZED ANTIBODY

Structural features associated with the ability of a monoclonal antibo dy (mAb) to discriminate between protein variants are identified and e ngineered. The variants are the curaremimetic toxin alpha from Naja ni gricollis and erabutoxin a or b from Laticauda semifasciata, which dif fer from each other by 16 substitutions and one insertion. The neutral izing mAb M alpha 1 recognizes with high affinity a topographical epit ope on the surface of toxin alpha, but fails to recognize the erabutox ins although they possess most of the residues forming the presumed ep itope. Examinations of the toxin alpha and erabutoxin 3-D structures a nd molecular dynamics simulations reveal several differences between t he variants. In particular, the region involving the beta-turn 17-24 i s organized differently. Analysis of the differences found in this reg ion suggests that the insertion (or deletion) at position 18 of the va riant amino acid sequences is particularly important in determining th e differential cross-reactivity. To test this proposal, residue 18 was deleted in one erabutoxin using site-directed mutagenesis, and the bi ological properties of the resulting mutant were examined. We found th at full antigenicity was restored in the previously unrecognized varia nt. The implications of this finding are discussed.