Structural features associated with the ability of a monoclonal antibody (mAb) to discriminate between protein variants are identified and engineered. The variants are the curaremimetic toxin alpha from Naja nigricollis and erabutoxin a or b from Laticauda semifasciata, which differ from each other by 16 substitutions and one insertion. The neutralizing mAb M alpha 1 recognizes with high affinity a topographical epitope on the surface of toxin alpha, but fails to recognize the erabutoxins although they possess most of the residues forming the presumed epitope. Examinations of the toxin alpha and erabutoxin 3-D structures and molecular dynamics simulations reveal several differences between the variants. In particular, the region involving the beta-turn 17-24 is organized differently. Analysis of the differences found in this region suggest that the insertion (or deletion) at position 18 of the variant amino acid sequences is particularly important in determining the differential cross-reactivity. To test this proposal, residue 18 was deleted in one erabutoxin using site-directed mutagenesis, and the biological properties of the resulting mutant were examined. We found that full antigenicity was restored in the previously unrecognized variant. The implications of this finding are discussed.