Participation of residue F552 in domain III of the protective antigen in the biological activity of anthrax lethal toxin

The protective antigen (PA) component of anthrax toxin translocates the catalytic moieties lethal factor (LF) and edema factor (EF) into the cytosol. The proteolytically activated 63 kDa form of PA (PA63) has the ability to oligomerize and bind LF/EF. PA has four distinct domains performing specialized functions; whereas the function of domains I, II and IV has been well characterized, domain III has no known role in the biological activity of PA. Here we report the role of amino acid residues lining an exposed hydrophobic patch of domain III in the biological activity of PA. The residues Phe552, Phe554, lIe562, Leu566 and lle574 were individually substituted with alanine and the effect was studied. All mutant PA proteins except Phe552Ala were equally active as wild-type PA in exhibiting a toxic phenotype to J774A.1 cells in the presence of LF. Substitution of Ala for Phe552 reduced the ability of PA to intoxicate cells by more than 250-fold. However, Phe552Ala was equally active in receptor binding and susceptibility to trypsin and chymotrypsin as wild-type PA, the activities that have been shown to be essential for the biological activity of PA. This mutated PA protein had a decreased ability to bind LF, oligomerize on cells and to induce release of 86Rb+ from Chinese hamster ovary cells. These results suggest that the residue Phe552 in PA plays an important role in LF binding and oligomerization. Our study provides a basis for further exploration of the biological significance of domain III of PA.     

Proteolytic stability of beta-peptide bonds probed using quenched fluorescent substrates incorporating a hemoglobin cleavage site

Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA₆₃, a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into the cell. Site-directed mutagenesis was used to substitute cysteine for each of three residues (N209, E614 and E733) at various levels on the lateral face of the PA₆₃ heptamer and for one residue (E126) on LF_N, the 30 kDa N-terminal PA binding domain of LF. Cysteine residues in PA were labeled with IAEDANS and that in LF_N was labeled with Alexa 488 maleimide. The mutagenesis and labeling did not significantly affect function. Time-resolved fluorescence methods were used to study fluorescence resonance energy transfer (FRET) between the AEDANS and Alexa 488 probes after the complex assembled in solution. The results clearly indicate energy transfer between AEDANS labeled at residue N209C on PA and the Alexa 488-labeled LF_N, whereas transfer from residue E614C on PA was slight, and none was observed from residue E733C. These results support a model in which LF_N binds near the top of the ring-shaped (PA₆₃)₇ heptamer.     

Mutagenesis and characterization of specific residues in fatty acid ethyl ester synthase: A gene for alcohol-induced cardiomyopathy

Fatty acid ethyl ester synthase-III metabolizes both ethanol and carcinogens. Structure-function studies of the enzyme have not been performed in relation to site specific mutagenesis. In this study, three residues (Gly 32, Cys 39 and His 72) have been mutated to observe their role in enzyme activity. Gly to Gln, Cys to Trp and His to Ser mutations did not affect fatty acid ethyl ester synthase activity, but His to Ser mutant had less than 9% of control glutathione S-transferase activity. The apparent loss of transferase activity reflected a 28 fold weaker binding constant for glutathione. Thus, this study indicates that Gly and Cys may not be important for synthase or transferase activities however, histidine may play a role in glutathione binding, but it is not an essential catalytic residue of glutathione S-transferase or for fatty acid ethyl ester synthase activity.     

Charge requirements for proton gradient-driven translocation of anthrax toxin

Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (ΔpH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ΔpH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ΔpH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LF(N)) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LF(N) to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LF(N) translocate independently of the ΔpH driving force. Acidic residues markedly increase the rate of ΔpH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ΔpH-driven unfolding and translocation. We discuss a ΔpH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the "molecular teeth" of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ΔpH translocation.     

Involvement of residues 147VYYEIGK153 in binding of lethal factor to protective antigen of Bacillus anthracis

Anthrax toxin is a complex of protective antigen (PA, 735 aa), lethal factor (LF, 776 aa), and edema factor (EF, 767 aa). PA binds to cell surface receptors and is cleaved by cell surface proteases into PA63, while LF and EF compete for binding to PA63. The PA63-LF/EF complex is internalized into the cytosol and causes different pathogenic responses in animals and cultured cells. 1-300 amino acid residues of LF have been viewed as the region responsible for the high affinity binding of LF to PA. Amino acid analysis of LF and EF revealed a common stretch of 7 amino acids (147VYYEIGK153). In the present study, each amino acid of this stretch was replaced by alanine at a time. Y148A, Y149A, I151A, and K153A mutants were found to be deficient in their ability to lyse J774A.1 cells and their binding ability to PA63 was drastically reduced. We propose that these four amino acids play a crucial role in the process of binding of LF to PA63.     

Anthrax lethal factor (LF) mediated block of the anthrax protective antigen (PA) ion channel: effect of ionic strength and voltage

The anthrax toxin complex consists of three different molecules, protective antigen (PA), lethal factor (LF), and edema factor (EF). The activated form of PA, PA(63), forms heptamers that insert at low pH in biological membranes forming ion channels and that are necessary to translocate EF and LF in the cell cytosol. LF and EF are intracellular active enzymes that inhibit the host immune system promoting bacterial outgrowth. Here, PA(63) was reconstituted into artificial lipid bilayer membranes and formed ion-permeable channels. The heptameric PA(63) channel contains a binding site for LF on the cis side of the channel. Full-size LF was found to block the PA(63) channel in a dose- and ionic-strength-dependent way with half-saturation constants in the nanomolar concentration range. The binding curves suggest a 1:1 relationship between (PA(63))(7) and bound LF that blocks the channel. The presence of a His(6) tag at the N-terminal end of LF strongly increases the affinity of LF toward the PA(63) channel, indicating that the interaction between LF and the PA(63) channel occurs at the N terminus of the enzyme. The LF-mediated block of the PA(63)-induced membrane conductance is highly asymmetric with respect to the sign of the applied transmembrane potential. The result suggested that the PA(63) heptamers contain a high-affinity binding site for LF inside domain 1 or the channel vestibule and that the binding is ionic-strength-dependent.     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.     

Residue Histidine 669 Is Essential for the Catalytic Activity of Bacillus anthracis Lethal Factor

The lethal factor (LF) of Bacillus anthracis is a Zn²⁺-dependent metalloprotease which plays an important role in anthrax virulence. This study was aimed at identifying the histidine residues that are essential to the catalytic activities of LF. The site-directed mutagenesis was employed to replace the 10 histidine residues in domains II, III, and IV of LF with alanine residues, respectively. The cytotoxicity of these mutants was tested, and the results revealed that the alanine substitution for His-669 completely abolished toxicity to the lethal toxin (LT)-sensitive RAW264.7 cells. The reason for the toxicity loss was further explored. The zinc content of this LF mutant was the same as that of the wild type. Also this LF mutant retained its protective antigan (PA)-binding activity. Finally, the catalytic cleavage activity of this mutant was demonstrated to be drastically reduced. Thus, we conclude that residue His-669 is crucial to the proteolytic activity of LF.Anthrax is a zoonotic disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis (24). Because infections are highly fatal, the organisms are easily produced, and the spores spread easily, B. anthracis has been used as a bioweapon in biological war and biological terrorism (38). If inhaled, the spores are phagocytosed by alveolar macrophages, where they germinate to produce vegetative bacteria (10, 24). The vegetative bacteria further release anthrax toxins, which inhibit the innate and adaptive immune responses of the hosts. This enables the capsulated bacteria to escape the lymph node defense barrier to reach the blood system, causing bacteremia and toxemia, which can rapidly kill the hosts (24, 26). The great threat posed by anthrax to the public is not only due to the highly lethal rate of inhaled anthrax, but also is due to the social panic caused by the lethality. Therefore, efficient ways to defend against anthrax infection and spreading are greatly needed. This mostly depends on a full understanding of the mechanisms of anthrax infection and toxicities.Anthrax toxins are the dominant virulence factors of Bacillus anthracis (6, 33, 37). They consist of three proteins: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and edema factor (EF; 89 kDa). The 83-kDa PA (PA₈₃) directly binds to cellular membrane receptors and was cleaved to an active fragment of 63-kDa PA (PA₆₃) by cellular proteases of the furin family or by serum proteases. The receptor-bound portion of PA₆₃ self-assembles into either ring-shaped heptamers, which bind to three molecules of LF and/or EF, resulting in (PA₆₃)₇(LF/EF)₃ (21), or octamers which bind up to four molecules of these moieties, resulting in (PA₆₃)₈(LF/EF)₄ complexes (16, 17). The catalytic partners (EF and/or LF) are subsequently transported across the membrane to the cell cytosol (24, 27). EF is a Ca²⁺- and calmodulin-dependent adenylate cyclase that, together with PA, forms edema toxin. EF causes a rapid increase in intracellular cyclic AMP (cAMP) levels in host cells and alters the elaborate balance of intracellular signaling pathways (20, 23). LF is a Zn²⁺-dependent protease that, together with PA, forms lethal toxin (LT). It is a dominant virulence factor and the major cause of death for the B. anthracis-infected animals (1, 29, 30). LF specifically cleaves the N-terminal domain of mitogen-activated protein kinase kinases (MAPKKs) (11, 35). Because the N-terminal domain of MAPKKs is essential for the interaction between MAPKKs and MAPKs, the cleavage of this domain impairs the activation of MAPKs (8, 11, 15) and leads to the inhibition of three major cellular signaling pathways—the ERK (extracellular signal-regulated kinase), p38, and JNK (c-Jun N-terminal kinase) pathways (29, 31)—and thus induces the lysis of the host cells in an unknown mechanism.The crystal structure of LF with the N-terminal domain of MEK2 has been reported (28). LF has 776 amino acids and comprises four different domains. Domain I (residues 1 to 254) is a PA-binding domain which delivers the remaining domains of the LF to the cell cytoplasm (3). The interface among domains II, III, and IV creates long, deep, 40-Å-long catalytic grooves into which the N terminus of MEK fits and forms an active site complex (28). Domain IV is central to catalytic activities of LF, containing two zinc-binding motifs (residues 686 to 690 and residues E735 to E739) and bound to a single Zn ion (18). However, which residues of LF are critical for efficient catalytic activities and execute the substrate cleavage remains unclear.Histidine is the only naturally occurring amino acid to contain an imidazole residue as a side chain. The catalytic activity of histidine mostly depends on the special features of the imidazole residue. The logarithm of the proton dissociation constant of imidazolyl in the histidine residue is about 6.5; thus, under the physiological condition, it tends to form hydrogen bonds and shares donor and acceptor properties that can take part in either nucleophilic or base catalysis. The speed of the imidazole residue to give or accept protons is very fast, with a half-life of less than 10 s. So in the process of natural selection, histidine was chosen as the catalytic structure, indicating that it plays an important role in the catalysis process of enzymes (9, 12, 14). There are 21 histidines in LF, with 9 of them in LF domain I and 12 of them in domains II, III, and IV. The histidine residues important to LF activities in domain I have been identified (2, 22). The other 12 histidine residues in the remaining three domains include His-277, His-280, and His-424 in domain II; His-309 in domain III; and His-588, His-645, His-654, His-669, His-686, His-690, His-745, and His-749 in domain IV (28). His-686 and His-690 in domain IV were demonstrated to form a zinc binding site constituting a thermolysin-like zinc metalloprotease motif, HEXXH (18). The activities of the remaining 10 histidine residues in domains II, III, and IV have not been explored yet. In this study, we replaced these 10 histidine residues separately with alanine residues by site-directed mutagenesis. By the cytotoxicity assay of all these mutants, the H669A mutant was found to lose cell toxicity completely. Further assay revealed that residue His-669 was involved in neither zinc stabilization nor PA binding but participated in the substrate proteolytic activity of LF.     

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional ¹³C‐edited ¹H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.     

Effect of pH on stability of anthrax lethal factor: correlation between denaturation and activity

Anthrax is caused by Gram positive bacterium Bacillus anthracis. Pathogenesis is result of production of three protein components, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA in combination with LF (lethal toxin) is lethal to animals, while PA in combination with EF (edema toxin), causes edema. PA, LF, and EF are very thermolabile. Differential scanning calorimetry (DSC) was used to unravel the energetics of LF denaturation as a function of pH ranging from 7.8 to 5.5. Transition temperature (T(m)) of LF was found to be approximately equal to 42 degrees C and onset of denaturation occurs at approximately equal to 30 degrees C. The ratio of calorimetric to van't Hoff's enthalpy was nearly equal to unity at pH 7.0, indicative of presence of single structural domain in LF at pH 7.0, unlike PA which has been structurally observed to consist of 4 domains. It was found by cytotoxicity studies using J774A.1 macrophage like cells that LF was most stable at pH approximately 6.5. This paper reports for the first time the denaturation of LF at different pH values at 37 degrees C and tries to establish a correlation between denaturation and loss of LF activity at different pH values.     

Trp 346 and Leu 352 residues in protective antigen are required for the expression of anthrax lethal toxin activity

The three separate proteins that make up anthrax toxin-protective antigen (PA), edema factor (EF) and lethal factor (LF) act in binary combinations to produce two distinct reactions in experimental animals: edema (PA+EF) and death (PA+LF). PA is believed to interact with a membrane receptor and, after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol. Residues W346, M350, and L352 in loop 3 of domain 2 have been implicated to induce a conformational change when the pH is lowered from 7.4 to 6.5. Modification of the residues Trp (346), Met (350), and Leu (352) to alanine individually and all the three residues together to alanine residues resulted in the loss of cytotoxic activity in combination with LF. The mutant proteins were able to bind to the cell surface receptor, become cleaved by trypsin, bind LF, and oligomerize. These residues might play an important role in the membrane insertion of PA and/or translocation of LF/EF into the cytosol.     

Mapping the anthrax protective antigen binding site on the lethal and edema factors.

Entry of anthrax edema factor (EF) and lethal factor (LF) into the cytosol of eukaryotic cells depends on their ability to translocate across the endosomal membrane in the presence of anthrax protective antigen (PA). Here we report attributes of the N-terminal domains of EF and LF (EF(N) and LF(N), respectively) that are critical for their initial interaction with PA. We found that deletion of the first 36 residues of LF(N) had no effect on its binding to PA or its ability to be translocated. To map the binding site for PA, we used the three-dimensional structure of LF and sequence similarity between EF and LF to select positions for mutagenesis. We identified seven sites in LF(N) (Asp-182, Asp-187, Leu-188, Tyr-223, His-229, Leu-235, and Tyr-236) where mutation to Ala produced significant binding defects, with H229A and Y236A almost completely eliminating binding. Homologous mutants of EF(N) displayed nearly identical defects. Cytotoxicity assays confirmed that the LF(N) mutations impact intoxication. The seven mutation-sensitive amino acids are clustered on the surface of LF and form a small convoluted patch with both hydrophobic and hydrophilic character. We propose that this patch constitutes the recognition site for PA.     

The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity.

Anthrax toxin consists of three separate proteins produced by Bacillus anthracis: protective antigen (PA), lethal factor (LF), and edema factor (EF). Previous work showed that the process by which these proteins damage eukaryotic cells begins with binding of PA (83 kDa) to cell surface receptors. PA is then cleaved by a cell surface protease so as to expose a high-affinity binding site for LF or EF on the COOH-terminal, receptor-bound, 63-kilodalton fragment. In this report we more closely define a region of PA involved in receptor binding. The gene encoding PA was mutagenized so as to delete 3, 5, 7, 12, or 14 amino acids from the carboxyl terminus of the protein, and the truncated PA variants were purified from Bacillus subtilis or Escherichia coli. Deletion of 3, 5, or 7 amino acids reduced the binding of PA to cells and the subsequent toxicity of the PA.LF complex to J774A.1 cells and also the ability to cause EF binding to cells. Deletion of 12 or 14 amino acids completely eliminated all these activities. These results show that the carboxy terminus comprises or is part of the receptor-binding domain of PA.     

Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis

Linker insertion mutagenesis was employed to create structural disruptions of the lethal factor (LF) protein of anthrax toxin to map functional domains. A dodecameric linker was inserted at 17 blunt end restriction enzyme sites throughout the gene. Paired MluI restriction sites within the linker allowed the inserts to be reduced from four to two amino acids. Shuttle vectors containing the mutated genes were transformed into the avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the gene products. Mutations at five sites in the central one-third of the sequence made the protein unstable, and purified protein could not be obtained. Mutated LF proteins with insertions at the other sites were purified and assessed for toxic activity in a macrophage lysis assay and for their ability to bind to the protective antigen (PA) component of anthrax toxin, the receptor binding moiety. Most insertions located in the NH2-terminal one-third of the LF protein eliminated both toxicity and binding to PA, while all four insertions in the COOH-terminal one-third of the protein eliminated toxicity without affecting binding to PA. These data support the hypothesis that the NH2-terminal domain contains the structures required for binding to PA and the COOH-terminal domain contains the putative catalytic domain of LF.     

血红素氧合酶HugZ组氨酸-249对组氨酸-245侧链缺失补偿的结构基础

血红素氧合酶HugZ是幽门螺旋杆菌(Helicobacter pylori)利用宿主血红素作为铁源的关键蛋白．HugZ的His245残基侧链咪唑基与血红素中心铁配位结合,是酶活中心的重要组成部分．用定点突变的方法构建HugZ突变体H245A、H249A和H245A/H249A基因,并将突变体蛋白表达纯化．通过X射线晶体学途径解析了突变体H245A与血红素复合物的2.55Å分辨率晶体结构．结构解析表明,HugZ的His249残基侧链咪唑基团与血红素的铁原子结合,从而补偿了His245侧链缺失．这种结构特征在已知血红素氧合酶中未曾发现．Val238 ψ平面的可翻转和Gly239的柔性是His249能与血红素配位结合的关键原因,二者的共同作用改变了C端肽链的走向,使Val238与His249之间的柔性回折与α1螺旋的相互作用发生解离,并向远离血红素的方向伸展．HugZ蛋白与血红素结合的光谱实验证明HugZ柔性C端上的组氨酸残基有利于HugZ与血红素的结合．研究结果表明,含多个组氨酸残基柔性C端的存在有利于血红素氧合酶HugZ结合和分解血红素．     

Large-scale structural changes accompany binding of lethal factor to anthrax protective antigen: a cryo-electron microscopic study

Anthrax toxin (AT), secreted by Bacillus anthracis, is a three-protein cocktail of lethal factor (LF, 90 kDa), edema factor (EF, 89 kDa), and the protective antigen (PA, 83 kDa). Steps in anthrax toxicity involve (1) binding of ligand (EF/LF) to a heptamer of PA63 (PA63h) generated after N-terminal proteolytic cleavage of PA and, (2) following endocytosis of the complex, translocation of the ligand into the cytosol by an as yet unknown mechanism. The PA63h.LF complex was directly visualized from analysis of images of specimens suspended in vitrified buffer by cryo-electron microscopy, which revealed that the LF molecule, localized to the nonmembrane-interacting face of the oligomer, interacts with four successive PA63 monomers and partially unravels the heptamer, thereby widening the central lumen. The observed structural reorganization in PA63h likely facilitates the passage of the large 90 kDa LF molecule through the lumen en route to its eventual delivery across the membrane bilayer.     

Structural determinants for the binding of anthrax lethal factor to oligomeric protective antigen

Anthrax lethal toxin assembles at the surface of mammalian cells when the lethal factor (LF) binds via its amino-terminal domain, LF(N), to oligomeric forms of activated protective antigen (PA). LF x PA complexes are then trafficked to acidified endosomes, where PA forms heptameric pores in the bounding membrane and LF translocates through these pores to the cytosol. We used enhanced peptide amide hydrogen/deuterium exchange mass spectrometry and directed mutagenesis to define the surface on LF(N) that interacts with PA. A continuous surface encompassing one face of LF(N) became protected from deuterium exchange when LF(N) was bound to a PA dimer. Directed mutational analysis demonstrated that residues within this surface on LF(N) interact with Lys-197 on two PA subunits simultaneously, thereby showing that LF(N) spans the PA subunit:subunit interface and explaining why heptameric PA binds a maximum of three LF(N) molecules. Our results elucidate the structural basis for anthrax lethal toxin assembly and may be useful in developing drugs to block toxin action.     

Purified Bacillus anthracis lethal toxin complex formed in vitro and during infection exhibits functional and biological activity

Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA(63)), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA(63) pore (approximately 12 angstroms) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA(63). In this study, we found that purified (PA(63))(7)-LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA(63) heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogen-activated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA(63) pore. Complexes of (PA(63))(7)-LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.     

Computational insights into the interaction of the anthrax lethal factor with the N-terminal region of its substrates

The anthrax lethal factor is a zinc metalloprotease toxin secreted by Bacillus anthracis which cleaves at the N-terminal region of six mitogen activated protein kinase kinases (MEKs) in the cell. Additionally, it is known to cleave a nine residue peptide "LF10," 50-fold more efficiently than nine residues of MEK1. There is very little sequence similarity between the MEK N-termini, thus, it is unclear how the lethal factor can accommodate and cleave the diverse N-termini of the MEKs and whether there is a hierarchy in this interaction, as there is between LF10 and MEK1. To investigate this problem, we carried out multiple molecular dynamics simulations of the lethal factor with nine residues of each of the substrates. Our simulations reveal that like LF10, certain MEK substrates have residue compositions that favor beta-sheet formation with the lethal factor over others. The formation of this secondary structure maintains a catalytic conformation. Binding energetics using the MM-PBSA method was used to rank-order the substrates for their affinity to LF (K(M)). On the basis of the results, we conclude that the LF does not equally accommodate the MEK substrates and further predict that there will be differences between rates of cleavage among the nine residue MEK N-termini.     

Binding of N-terminal fragments of anthrax edema factor (EF(N)) and lethal factor (LF(N)) to the protective antigen pore

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA(63), forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA(63)-channels. Here, in experiments with artificial lipid bilayer membranes, EF(N) and LF(N) show block of PA(63)-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF(N), increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA(63)-channel.