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

M. J. Rosovitz, Peter Schuck, Mini Varughese, Arun P. Chopra, Varsha Mehra, Yogendra Singh, Lisa M. McGinnis, Stephen H. Leppla
2003 Journal of Biological Chemistry  
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 Asp 683 was the
more » ... t critical for cell binding and toxicity, causing an ϳ1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr 681 , Asn 682 , and Pro 686 also reduced toxicity significantly, by 10 -100-fold. Of these, only Asn 682 and Pro 686 were also critical for interactions with 14B7. However, residues Lys 684 , Leu 685 , Leu 687 , and Tyr 688 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. Bacillus anthracis secretes two toxins: edema toxin and lethal toxin. Each is composed of a common binding component, protective antigen (PA), 1 together with an enzymatic component, edema factor (EF), in the case of edema toxin and lethal factor (LF) in the case of lethal toxin (1-3). The current model for toxin entry into the cell illustrates the centrality of PA for toxin action. PA binds to cellular receptors, recently identified as splice variants of either tumor endothelial marker 8 (TEM8) (4 -6) or the closely related capillary morphogenesis protein 2 (CMG2) (7). Furin cleaves PA, releasing a 20-kDa fragment and leaving behind a 63-kDa portion (PA 63 ) capable of forming a heptamer, which has a newly exposed surface that binds EF and LF (8 -12). Heptamer complexes enter the endocytic pathway by receptor-mediated endocytosis (13), and upon acidification of the vesicle, the PA 63 heptamer undergoes a conforma-tional change to form a pore through which EF and LF translocate into the cytoplasm (10, 11, 14 -16). Once in the cytoplasm, EF and LF exert their toxic effects. The PA protein can be divided into four domains based on its crystal structure, and functions can be attributed to the different domains based on mutational and biochemical analyses (16). Domain 1 (residues 1-258) contains the furin cleavage site as well as the hydrophobic portion of PA, which is exposed upon furin cleavage to allow EF and LF to bind (16, 17). Several lines of evidence indicate that domain 2 (residues 259 -487) is involved in oligomerization and contains the loop that inserts into the membrane to form the channel through which the LF and EF enter the cytosol (16, 18 -20). Various amino acids in domain 3 (residues 488 -595) are necessary for oligomerization, and this has been the only function attributed to domain 3 to date (21, 22) . Domain 4 (residues 596 -735) is essential for binding to cellular receptor as indicated by several lines of evidence. A CNBr fragment of PA containing residues 663-735 successfully competed with PA in cell binding assays, and mutations in domain 4 prevented PA binding to cells (23-25). Monoclonal antibody and mutational analysis studies further localized the residues necessary for binding to receptor. Monoclonal antibody 14B7 neutralized anthrax toxin by inhibiting PA binding to receptor and recognized a region of PA between residues 671 and 721 (17). Additionally, multisubstituted PA variants in and near the small loop of domain 4 (between ␤ strands 4␤ 8 and 4␤ 9 ) suggested that residues essential for binding were in that region (25). The alanine substitutions described in this work cover the small loop (amino acids 679 -693) of domain 4. We also included another residue, Asn 657 , located nearby in the crystal structure and implicated by our earlier multisubstituted variants. The development of new reagents to prevent and treat anthrax can be aided by our understanding of the molecular interactions between the toxin components and cellular targets as well as by interactions between the toxin components and potential reagents. One of the molecular reactions it would be beneficial to understand in molecular detail would be that between anthrax toxin and its cellular receptor. As noted above, much progress has been made in the study of PA and recently also with its cellular receptors, TEM8 and capillary morphogenesis protein 2, but studies of the interaction between the toxin with receptor have to date been limited to deletions or multiple substitutions in the proteins (6, 25). One goal of this study was to determine which individual amino acids in the previously identified region of PA were important for interaction with its receptor. For development of improved vaccines and antibody-based
doi:10.1074/jbc.m301154200 pmid:12771151 fatcat:h2gvpyf2engg7pibg23nmrli5u