title: 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 approximately 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.
authors.affiliation: Microbial Pathogenesis Section, NIAID, National Institutes of Health, Bethesda, Maryland 20892-4350, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Rosovitz
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
authors.forename: Mini
authors.initials: M
authors.lastname: Varughese
authors.forename: Arun P
authors.initials: AP
authors.lastname: Chopra
authors.forename: Varsha
authors.initials: V
authors.lastname: Mehra
authors.forename: Yogendra
authors.initials: Y
authors.lastname: Singh
authors.forename: Lisa M
authors.initials: LM
authors.lastname: McGinnis
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Leppla
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000942:Antigens, Bacterial; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites / Q000276:immunology; D002460:Cell Line; D008264:Macrophages / Q000166:cytology / Q000276:immunology* / Q000382:microbiology; D051379:Mice; D008956:Models, Chemical; D009154:Mutation; D017434:Protein Structure, Tertiary; D018000:Receptors, Peptide / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D018000:Receptors, Peptide; C030325:anthrax toxin; C070309:anthrax toxin receptors; D001224:Aspartic Acid; D000409:Alanine