The Structure of Tumor Endothelial Marker 8 (TEM8) Extracellular Domain and Implications for Its Receptor Function for Recognizing Anthrax Toxin

Anthrax toxin, which is released from the Gram-positive bacterium Bacillus anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds a receptor on the surface of the target cell and further assembles into a homo-heptameric pore through which EF and LF translocate into the cytosol. Two distinct cellular receptors for anthrax toxin, TEM8/ANTXR1 and CMG2/ANTXR2, have been identified, and it is known that their extracellular domains bind PA with low and high affinities, respectively. Here, we report the crystal structure of the TEM8 extracellular vWA domain at 1.7 Å resolution. The overall structure has a typical integrin fold and is similar to that of the previously published CMG2 structure. In addition, using structure-based mutagenesis, we demonstrate that the putative interface region of TEM8 with PA (consisting of residues 56, 57, and 154–160) is responsible for the PA-binding affinity differences between the two receptors. In particular, Leu56 was shown to be a key factor for the lower affinity of TEM8 towards PA compared with CMG2. Because of its high affinity for PA and low expression in normal tissues, an isolated extracellular vWA domain of the L56A TEM8 variant may serve as a potent antitoxin and a potential therapeutic treatment for anthrax infection. Moreover, as TEM8 is often over-expressed in tumor cells, our TEM8 crystal structure may provide new insights into how to design PA mutants that preferentially target tumor cells.     

Selection of anthrax toxin protective antigen variants that discriminate between the cellular receptors TEM8 and CMG2 and achieve targeting of tumor cells

Anthrax toxin, a three-component protein toxin secreted by Bacillus anthracis, assembles into toxic complexes at the surface of receptor-bearing eukaryotic cells. The protective antigen (PA) protein binds to receptors, either tumor endothelial cell marker 8 (TEM8) or CMG2 (capillary morphogenesis protein 2), and orchestrates the delivery of the lethal and edema factors into the cytosol. TEM8 is reported to be overexpressed during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues. To extend prior work on targeting of tumor with modified anthrax toxins, we used phage display to select PA variants that preferentially bind to TEM8 as compared with CMG2. Substitutions were randomly introduced into residues 605-729 of PA, within the C-terminal domain 4 of PA, which is the principal region that contacts receptor. Candidates were characterized in cellular cytotoxicity assays with Chinese hamster ovary (CHO) cells expressing either TEM8 or CMG2. A PA mutant having the substitutions R659S and M662R had enhanced specificity toward TEM8-overexpressing CHO cells. This PA variant also displayed broad and potent tumoricidal activity to various human tumor cells, especially to HeLa and A549/ATCC cells. By contrast, the substitution N657Q significantly reduced toxicity to TEM8 but not CMG2-overexpressing CHO cells. Our results indicate that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.     

Characterization of the interaction between anthrax toxin and its cellular receptors

Mutations in capillary morphogenesis gene 2 (CMG2), one of the two closely related proteins that act as anthrax toxin receptors, cause two rare human autosomal recessive conditions, juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH). Here we demonstrate that CMG2 proteins with certain JHF- and ISH-associated single amino acid substitutions in their von Willebrand factor A domain or transmembrane region do not function as anthrax toxin receptors. However, an ISH-associated CMG2 variant having a truncated cytosolic domain does still function as an anthrax receptor, and in fact makes cells hyper-sensitive to toxin, distinguishing the roles of CMG2 in physiology and anthrax pathology. Site-specific mutagenesis was used to characterize the role that domain 2 of the anthrax toxin protective antigen (PA) plays in interaction with CMG2, focusing on the interaction between the PA 2beta(3)-2beta(4) loop and a pocket (Glu-122 pocket) adjacent to the metal ion-dependent adhesion site in CMG2. Substitutions that disrupted the salt bridge between PA Arg-344 and CMG2 Glu-122 decreased the affinity of PA to CMG2 three- to fourfold. Furthermore, mutation of CMG2 Tyr-119 (within the Glu-122 pocket) to His lowered the pH threshold for PA prepore-to-pore conversion in the endocytic pathway.     

Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen

The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates entry of the two enzymatic moieties of the toxin into the cytosol. Two PA receptors, anthrax toxin receptor (ATR)/tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been identified. We expressed and purified the von Willebrand A (VWA) domain of CMG2 and examined its interactions with monomeric and heptameric forms of PA. Monomeric PA bound a stoichiometric equivalent of CMG2, whereas the heptameric prepore form bound 7 eq. The Kd of the VWA domain-PA interaction is 170 pm when liganded by Mg2+, reflecting a 1000-fold tighter interaction than most VWA domains with their endogenous ligands. The dissociation rate constant is extremely slow, indicating a 30-h lifetime for the CMG2.PA monomer complex. CMG2 metal ion-dependent adhesion site (MIDAS) was studied kinetically and thermodynamically. The association rate constant (approximately 10(5) m(-1) s(-1)) is virtually identical in the presence or absence of Mg2+ or Ca2+ , but the dissociation rate of metal ion liganded complex is up to 4 orders of magnitude slower than metal ion free complex. Residual affinity (Kd approximately 960 nm) in the absence of divalent metal ions allowed the free energy for the contribution of the metal ion to be calculated as 5 kcal mol(-1), demonstrating that the metal ion-dependent adhesion site is directly coordinated by CMG2 and PA in the binding interface. The high affinity of the VWA domain for PA supports its potency in neutralizing anthrax toxin, demonstrating its potential utility as a novel therapeutic for anthrax.     

A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein

Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.     

The inhibition of the interaction between the anthrax toxin and its cellular receptor by an anti-receptor monoclonal antibody

The high affinity binding of the anthrax protective antigen (PA) to one of its receptors, capillary morphogenesis protein 2 (CMG2), is essential for the intoxication process of anthrax toxin. To acquire novel research tools to study the PA-CMG2 interaction, we generated several anti-CMG2 monoclonal antibodies (MAbs). We demonstrated that one of the MAbs, 4B5, could inhibit PA-CMG2 binding and could also protect the sensitive cells against an anthrax lethal toxin challenge. We identified the epitope recognized by 4B5 and confirmed that the key residues of the epitope were the residues ¹¹⁹YI-LK¹²⁵ of CMG2. Based on our results, we propose that 4B5 binds to the E122 pocket of CMG2 and interrupts the interaction between the pocket and the PA 2β3-2β4 loop. To our knowledge, this is the first report to illustrate that an anti-CMG2 antibody could inhibit the PA-CMG2 interaction and therefore interfere with the intoxication of anthrax toxin.     

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.     

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a β-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.     

Endocytosis of the Anthrax Toxin Is Mediated by Clathrin,Actin and Unconventional Adaptors

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin.     

Mutations in the gene encoding capillary morphogenesis protein 2 cause juvenile hyaline fibromatosis and infantile systemic hyalinosis

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive conditions characterized by multiple subcutaneous skin nodules, gingival hypertrophy, joint contractures, and hyaline deposition. We previously mapped the gene for JHF to chromosome 4q21. We now report the identification of 15 different mutations in the gene encoding capillary morphogenesis protein 2 (CMG2) in 17 families with JHF or ISH. CMG2 is a transmembrane protein that is induced during capillary morphogenesis and that binds laminin and collagen IV via a von Willebrand factor type A (vWA) domain. Of interest, CMG2 also functions as a cellular receptor for anthrax toxin. Preliminary genotype-phenotype analyses suggest that abrogation of binding by the vWA domain results in severe disease typical of ISH, whereas in-frame mutations affecting a novel, highly conserved cytoplasmic domain result in a milder phenotype. These data (1) demonstrate that JHF and ISH are allelic conditions and (2) implicate perturbation of basement-membrane matrix assembly as the cause of the characteristic perivascular hyaline deposition seen in these conditions.     

The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin

Toxins produced by Bacillus anthracis and other microbial pathogens require functions of host cell genes to yield toxic effects. Here we show that low density lipoprotein receptor-related protein 6 (LRP6), previously known to be a coreceptor for the Wnt signaling pathway, is required for anthrax toxin lethality in mammalian cells. Downregulation of LRP6 or coexpression of a truncated LRP6 dominant-negative peptide inhibited cellular uptake of complexes containing the protective antigen (PA) carrier of anthrax toxin moieties and protected targeted cells from death, as did antibodies against epitopes in the LRP6 extracellular domain. Fluorescence microscopy and biochemical analyses showed that LRP6 enables toxin internalization by interacting at the cell surface with PA receptors TEM8/ATR and/or CMG2 to form a multicomponent complex that enters cells upon PA binding. Our results, which reveal a previously unsuspected biological role for LRP6, identify LRP6 as a potential target for countermeasures against anthrax toxin lethality.     

Improving the Anti-Toxin Abilities of the CMG2-Fc Fusion Protein with the Aid of Computational Design

CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.     

Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding,proteolytic processing,oligomer formation,and internalization

The interaction of anthrax toxin protective antigen (PA) and target cells was assessed, and the importance of the cytosolic domain of tumor endothelium marker 8 (TEM8) in its function as a cellular receptor for PA was evaluated. PA binding and proteolytic processing on the Chinese hamster ovary cell surface occurred rapidly, with both processes nearly reaching steady state in 5 min. Remarkably, the resulting PA63 fragment was present on the cell surface only as an oligomer, and furthermore, the oligomer was the only PA species internalized, suggesting that oligomerization of PA63 triggers receptor-mediated endocytosis. Following internalization, the PA63 oligomer was rapidly and irreversibly transformed to an SDS/heat-resistant form, in a process requiring an acidic compartment. This conformational change was functionally correlated with membrane insertion, channel formation, and translocation of lethal factor into the cytosol. To explore the role of the TEM8 cytosolic tail, a series of truncated TEM8 mutants was transfected into a PA receptor-deficient Chinese hamster ovary cell line. Interestingly, all of the cytosolic tail truncated TEM8 mutants functioned as PA receptors, as determined by PA binding, processing, oligomer formation, and translocation of an lethal factor fusion toxin into the cytosol. Moreover, cells transfected with a TEM8 construct truncated before the predicted transmembrane domain failed to bind PA, demonstrating that residues 321-343 are needed for cell surface anchoring. Further evidence that the cytosolic domain plays no essential role in anthrax toxin action was obtained by showing that TEM8 anchored by a glycosylphosphatidylinositol tail also functioned as a PA receptor.     

Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.     

Crystal Structure of the Engineered Neutralizing Antibody M18 Complexed to Domain 4 of the Anthrax Protective Antigen

The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity, single-chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here, we report the high-resolution X-ray structures of three high-affinity, single-chain antibodies in the 14B7 family; 14B7 and two high-affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 Å resolution. These structures provide insights into the mechanism of neutralization, and the effect of various mutations on antibody affinity, and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.     

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.     

Stability of domain 4 of the anthrax toxin protective antigen and the effect of the VWA domain of CMG2 on stability

The major immunogenic component of the current anthrax vaccine, anthrax vaccine adsorbed (AVA) is protective antigen (PA). We have shown recently that the thermodynamic stability of PA can be significantly improved by binding to the Von‐Willebrand factor A (VWA) domain of capillary morphogenesis protein 2 (CMG2), and improvements in thermodynamic stability may improve storage and long‐term stability of PA for use as a vaccine. In order to understand the origin of this increase in stability, we have isolated the receptor binding domain of PA, domain 4 (D4), and have studied the effect of the addition of CMG2 on thermodynamic stability. We are able to determine a binding affinity between D4 and CMG2 (～300 nM), which is significantly weaker than that between full‐length PA and CMG2 (170–300 pM). Unlike full‐length PA, we observe very little change in stability of D4 on binding to CMG2, using either fluorescence or ¹⁹F‐NMR experiments. Because in previous experiments we could observe a stabilization of both domain 4 and domain 2, the mechanism of stabilization of PA by CMG2 is likely to involve a mutual stabilization of these two domains.     

Whole-cell voltage clamp measurements of anthrax toxin pore current

Protective antigen (PA) of anthrax toxin binds cellular receptors and forms pores in target cell membranes, through which catalytic lethal factor (LF) and edema factor (EF) are believed to translocate to the cytoplasm. Using patch clamp electrophysiological techniques, we assayed pore formation by PA in real time on the surface of cultured cells. The membranes of CHO-K1 cells treated with activated PA had little to no electrical conductivity at neutral pH (7.3) but exhibited robust mixed ionic currents in response to voltage stimuli at pH 5.3. Pore formation depended on specific cellular receptors and exhibited voltage-dependent inactivation at large potentials (>60 mV). The pH requirement for pore formation was receptor-specific as membrane insertion occurs at significantly different pH values when measured in cells specifically expressing tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), the two known cellular receptors for anthrax toxin. Pores were inhibited by an N-terminal fragment of LF and by micromolar concentrations of tetrabutylammonium ions. These studies demonstrated basic biophysical properties of PA pores in cell membranes and served as a foundation for the study of LF and EF translocation in vivo.     

Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein

Background

To initiate infection, Bacillus anthracis needs to overcome the host innate immune system. Anthrax toxin, a major virulence factor of B. anthracis, impairs both the innate and adaptive immune systems and is important in the establishment of anthrax infections.

Methodology/Principal Findings

To measure the ability of anthrax toxin to target immune cells, studies were performed using a fusion of the anthrax toxin lethal factor (LF) N-terminal domain (LFn, aa 1–254) with β-lactamase (LFnBLA). This protein reports on the ability of the anthrax toxin protective antigen (PA) to mediate LF delivery into cells. Primary immune cells prepared from mouse spleens were used in conjunction with flow cytometry to assess cleavage and resulting FRET disruption of a fluorescent β-lactamase substrate, CCF2/AM. In spleen cell suspensions, the macrophages, dendritic cells, and B cells showed about 75% FRET disruption of CCF2/AM due to cleavage by the PA–delivered LFnBLA. LFnBLA delivery into CD4+ and CD8+ T cells was lower, with 40% FRET disruption. When the analyses were done on purified samples of individual cell types, similar results were obtained, with T cells again having lower LFnBLA delivery than macrophages, dendritic cells, and B cells. Relative expression levels of the toxin receptors CMG2 and TEM8 on these cells were determined by real-time PCR. Expression of CMG2 was about 1.5-fold higher in CD8+ cells than in CD4+ and B cells, and 2.5-fold higher than in macrophages.

Conclusions/Significance

Anthrax toxin entry and activity differs among immune cells. Macrophages, dendritic cells, and B cells displayed higher LFnBLA activity than CD4+ and CD8+ T cells in both spleen cell suspension and the purified samples of individual cell types. Expression of anthrax toxin receptor CMG2 is higher in CD4+ and CD8+ T cells, which is not correlated to the intracellular LFnBLA activity.     

Expression and purification of functional human anthrax toxin receptor (ATR/TEM8) binding domain from Escherichia coli

Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. Anthrax receptors play a crucial role in the pathogenesis of the anthrax disease. Anthrax toxin receptor ATR/TEM8 VWA domain is responsible for the binding of protective antigen (PA) of B. anthracis, and thus an attractive target for structure-based drug therapies. However, the production of soluble and functional ATR/TEM8 VWA domain currently requires the use of mammalian expression systems. In this work, we expressed the ATR/TEM8 VWA domain as a fusion protein in Escherichia coli. Recombinant ATR/TEM8 VWA domain has been purified to homogeneity, and its identity has been verified by both N-terminal protein microsequencing and mass spectrometry. The purified ATR/TEM8 VWA domain exhibits very high affinity to PA based on BIAcore assay. Moreover, like the domain expressed in mammalian system, the bacterially expressed ATR/TEM8 VWA domain can block cytotoxicity induced by anthrax toxins, suggesting that the bacterially expressed ATR/TEM8 VWA domain is properly folded and fully functional.