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 Asp 683 was the most 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 . Heptamer complexes enter the endocytic pathway by receptor-mediated endocytosis (13), and upon acidification of the vesicle, the PA 63 heptamer undergoes a conformational 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...

Structure of heptameric protective antigen bound to an anthrax toxin receptor: A role for receptor in pH-dependent pore formation

“…2b). Extensive site-directed mutagenesis in PA corroborates the importance of some of the domain 4 interface residues (I656, N682, and D683) in receptor binding and toxicity (15,16). Although a similar analysis has not yet been performed for CMG2, four of the CMG2 residues in the CMG2-PA interface (Y119, H121, E122, and Y158) are strictly conserved in ATR͞TEM8 and probably result in a common interaction with PA (Fig.…”

“…1b). Domain 4 (residues 596-735) has limited contact with the rest of PA and has been implicated by blocking antibodies, proteolysis, and mutagenesis to function in host-cell receptor binding (13)(14)(15)(16) (Fig. 1a).…”

Identification of Linear Epitopes in Bacillus anthracis Protective Antigen Bound by Neutralizing Antibodies

“…The boundary between domains 3 and 4, which does not have a known functional activity, has been suggested as a region recognized by polyclonal antibodies from vaccinated humans and rabbits (6,12). The cellular receptor binding region is localized to the small loop of domain 4, and this region has been described to be recognized by two neutralizing mAbs (7,9). With the exception of a neutralizing mAb that bound to PA 20 (13), no B-cell epitopes have been reported in domain 1.…”

“…The development of numerous monoclonal antibodies (mAbs) to different epitopes on the PA molecule that influence PA functions, in conjunction with epitope mapping, has provided an opportunity to study the fine antigenic structure of PA. Most of these epitopes have been defined in mice (5)(6)(7)(8), in macaques (9), in rabbits (10), as well as in vaccinated humans (11). Consequently, the epitopes described thus far are localized to three discrete regions within the PA.…”

Immunogenicity of Bacillus anthracis Protective Antigen Domains and Efficacy of Elicited Antibody Responses Depend on Host Genetic Background

“…It is known that antibodies to PA are essential for immunity to B. anthracis (10,14,16,20,25), and the binding sites of several monoclonal antibodies (MAbs) have been mapped to key domains on the PA molecule (21,36). Anthrax vaccine adsorbed is the current licensed anthrax vaccine for humans in the United States and consists of the cell-free culture filtrate of an attenuated strain of B. anthracis combined with an aluminum adjuvant (15,44).…”