Alanine-scanning mutations in domain 4 of anthrax toxin protective antigen reveal residues important for binding to the cellular receptor and to a neutralizing monoclonal antibody

Abstract

A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp⁶⁸³ was the most critical for cell binding and toxicity, causing an ~1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr⁶⁸¹, Asn⁶⁸², and Pro⁶⁸⁶ also reduced toxicity significantly, by 10-100-fold. Of these, only Asn⁶⁸² and Pro⁶⁸⁶ were also critical for interactions with 14B7. However, residues Lys⁶⁸⁴, Leu⁶⁸⁵, Leu⁶⁸⁷, and Tyr⁶⁸⁸ were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5- and 5-fold, respectively.