Model of the toxic complex of anthrax: responsive conformational changes in both the lethal factor and the protective antigen heptamer

The toxic complex of anthrax is formed when the monomeric protective antigen (PA) (83 kDa), while bound to its cell-surface receptor, is first converted to PA63 heptamers (PA63h) following N-terminal proteolytic cleavage, and then lethal (LF) (90 kDa) or edema factor (EF) binds to the heptamer. We report a "pseudoatomic" model for the complex of PA63h and full-length LF determined by applying the normal-mode flexible fitting procedure to a approximately 18 A cryo-electron microscopy (EM) density map of the complex. The model describes the interacting surface that buries a total area of approximately 10,140 A2 comprising approximately 40% charged, and approximately 30% each of polar and hydrophobic residues. For the heptamer, the buried surface, composed of approximately 110 residues, involves primarily three monomers and includes for two, similar stretches of the polypeptide chain from domain 1. For LF, the interface again involves approximately 110 residues, mostly from the N-terminal domain I (LF(N)), and the structurally homologous C-terminal domain IV. Most interestingly, bound LF displays a marked conformational change resulting from a "collapse" of domains I, III, and IV on domain II, with the largest movement of approximately 9 A noted for domain I. On the other hand, primarily, rigid-body movements, larger than approximately 10 A for three PA63 monomers, cause the hourglass-shaped heptamer lumen to enlarge by as much as approximately 50% near the middle of the molecule. Such concerted structural rearrangements in LF and the heptamer can facilitate ingress of the ligand into the heptamer lumen prior to unfolding and release through the PA63h channel formed in the acidic late endosomal membrane.     

Anthrax edema factor, voltage-dependent binding to the protective antigen ion channel and comparison to LF binding

Anthrax toxin complex 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 N-terminal part of PA, PA(63), forms a heptameric channel that inserts at low pH in endosomal membranes and that is necessary to translocate EF and LF in the cytosol of the target cells. EF is an intracellular active enzyme, which is a calmodulin-dependent adenylate cyclase (89 kDa) that causes a dramatic increase of intracellular cAMP level. Here, the binding of full-length EF on heptameric PA(63) channels was studied in experiments with artificial lipid bilayer membranes. Full-length EF blocks the PA(63) channels in a dose, temperature, voltage, and ionic strength-dependent way with half-saturation constants in the nanomolar concentration range. EF only blocked the PA(63) channels when PA(63) and EF were added to the same side of the membrane, the cis side. Decreasing ionic strength and increasing transmembrane voltage at the cis side of the membranes resulted in a strong decrease of the half-saturation constant for EF binding. This result suggests that ion-ion interactions are involved in EF binding to the PA heptamer. Increasing temperature resulted in increasing half-saturation constants for EF binding to the PA(63) channels. The binding characteristics of EF to the PA(63) channels are compared with those of LF binding. The comparison exhibits similarities but also remarkable differences between the bindings of both toxins to the PA(63) channel.     

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.     

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.     

Both PA63 and PA83 are endocytosed within an anthrax protective antigen mixed heptamer: a putative mechanism to overcome a furin deficiency

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.     

Anthrax toxin complexes: heptameric protective antigen can bind lethal factor and edema factor simultaneously

The 83 kDa protective antigen (PA(83)) component of anthrax toxin, after proteolytic activation, self-associates to form ring-shaped heptamers ([PA(63)](7)) that bind and aid delivery of the Edema Factor (EF) and Lethal Factor (LF) components to the cytosol. Here we show using fluorescence (F?rster) resonance energy transfer that a molecule of [PA(63)](7) can bind EF and LF simultaneously. We labeled EF and LF with an appropriate donor/acceptor pair and found quenching of the donor and an increase in sensitized emission of the acceptor when, and only when, a mixture of the labeled proteins was combined with [PA(63)](7). Addition of unlabeled PA(63)-binding domain of LF to the mixture competitively displaced labeled EF and LF, causing a loss of energy transfer. In view of the known maximum occupancy of 3 ligand molecules per [PA(63)](7), these findings indicate that PA, EF, and LF can form mixtures of liganded toxin complexes containing both EF and LF.     

Anthrax biosensor, protective antigen ion channel asymmetric blockade

The significant threat posed by biological agents (e.g. anthrax, tetanus, botulinum, and diphtheria toxins) (Inglesby, T. V., O'Toole, T., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Friedlander, A. M., Gerberding, J., Hauer, J., Hughes, J., McDade, J., Osterholm, M. T., Parker, G., Perl, T. M., Russell, P. K., and Tonat, K. (2002) J. Am. Med. Assoc. 287, 2236-2252) requires innovative technologies and approaches to understand the mechanisms of toxin action and to develop better therapies. Anthrax toxins are formed from three proteins secreted by fully virulent Bacillus anthracis, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). Here we present electrophysiological measurements demonstrating that full-length LF and EF convert the current-voltage relationship of the heptameric PA63 ion channel from slightly nonlinear to highly rectifying and diode-like at pH 6.6. This effect provides a novel method for characterizing functional toxin interactions. The method confirms that a previously well characterized PA63 monoclonal antibody, which neutralizes anthrax lethal toxin in animals in vivo and in vitro, prevents the binding of LF to the PA63 pore. The technique can also detect the presence of anthrax lethal toxin complex from plasma of infected animals. The latter two results suggest the potential application of PA63 nanopore-based biosensors in anthrax therapeutics and diagnostics.     

Anthrax protective antigen: efficiency of translocation is independent of the number of ligands bound to the prepore

Heptameric anthrax protective antigen (termed prepore), which assembles at the mammalian cell surface, competitively binds edema factor (EF) and/or lethal factor (LF). It then transports them to an acidic intracellular compartment and mediates their translocation across the membrane to the cytosol. Steric constraints limit to three the number of molecules of EF and/or LF that can bind simultaneously to prepore. To determine whether the number of ligand molecules bound per heptamer affects the efficiency of translocation, we measured the low-pH-triggered translocation of the radiolabeled protective antigen (PA(63))-binding domain of LF ((35)S-LF(N)) across the plasma membrane of CHO-K1 cells as a function of the degree of saturation of the prepore. The fraction translocated remained constant at approximately 0.4 as (35)S-LF(N) was varied from nil through saturating concentrations. The same constant value was observed when we held (35)S-LF(N) at a saturating concentration and varied the number of functional ligand sites per prepore by changing the ratio of wild-type PA to a ligand-binding mutant. Thus, prepore containing only a single ligand-binding site is capable of translocating its cargo as efficiently as one containing multiple binding sites. The results as a whole imply that heptamers with one, two, or three ligands bound translocate their ligands with the same efficiency, indicating that each ligand molecule is translocated independently from the others.     

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.     

Binding of N-terminal fragments of anthrax edema factor (EFN) and lethal factor (LFN) 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, PA₆₃, 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 PA₆₃-channels. Here, in experiments with artificial lipid bilayer membranes, EF_N and LF_N show block of PA₆₃-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 PA₆₃-channel.     

A quantitative study of the interactions of Bacillus anthracis edema factor and lethal factor with activated protective antigen

Bacillus anthracis secretes three proteins, which associate in binary combinations to form toxic complexes at the surface of mammalian cells. Receptor-bound protective antigen (PA) is proteolytically activated, yielding a 63 kDa fragment (PA(63)). PA(63) oligomerizes into heptamers, which bind edema factor (EF) or lethal factor (LF) to form the toxic complexes. We undertook a quantitative analysis of the interactions of EF with PA(63) by means of surface plasmon resonance (SPR) measurements. Heptameric PA(63) was covalently bound by amine coupling to an SPR chip, or noncovalently bound via a C-terminal hexahistidine tag on the protein to Ni(2+)nitrilotriacetate groups on the chip. Values of k(on) and k(off) for EF at 23 degrees C were approximately 3 x 10(5) M(-)(1) s(-)(1) and (3-5) x 10(-)(4) s(-)(1), respectively, giving a calculated K(d) of (1-2) x 10(-)(9) M. A similar value of K(d) (7 x 10(-)(10) M) was obtained when we measured the binding of radiolabeled EF to receptor-bound PA(63) on the surface of L6 cells (at 4 degrees C). Each of these analyses was also performed with LF and LF(N) (the N-terminal 255 residues of LF), and values obtained were comparable to those for EF. The similarity in the dissociation constants determined by SPR and by measurements on the cell surface suggests that the presence of the receptor does not play a large role in the interaction between PA(63) and EF/LF.     

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.     

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.     

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.     

Stoichiometry of anthrax toxin complexes.

After being proteolytically activated, the protective antigen (PA) moiety of anthrax toxin self-associates to form symmetric, ring-shaped heptamers. Heptameric PA competitively binds the enzymatic moieties of the toxin, edema factor and lethal factor, and translocates them across the endosomal membrane by a pH-dependent process. We used two independent approaches to determine how many of the seven identical EF/LF binding sites of the PA heptamer can be occupied simultaneously. We measured isotope ratios in complexes assembled from differentially radiolabeled toxin subunits, and we determined the molecular masses of unlabeled complexes by multiangle laser light scattering. Both approaches yielded the same value: the PA heptamer in solution binds three molecules of protein ligand under saturating conditions. This suggests that each bound ligand sterically occludes the binding sites of two PA subunits. According to this model, a ligand-saturated heptamer is asymmetric, with the sites of six of the seven subunits occluded. These results contribute to the conceptual framework for understanding the mechanism of membrane translocation by anthrax toxin.     

Anthrax toxin receptor drives protective antigen oligomerization and stabilizes the heptameric and octameric oligomer by a similar mechanism

Background

Anthrax toxin is comprised of protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins are individually nontoxic; however, when PA assembles with LF and EF, it produces lethal toxin and edema toxin, respectively. Assembly occurs either on cell surfaces or in plasma. In each milieu, PA assembles into a mixture of heptameric and octameric complexes that bind LF and EF. While octameric PA is the predominant form identified in plasma under physiological conditions (pH 7.4, 37°C), heptameric PA is more prevalent on cell surfaces. The difference between these two environments is that the anthrax toxin receptor (ANTXR) binds to PA on cell surfaces. It is known that the extracellular ANTXR domain serves to stabilize toxin complexes containing the PA heptamer by preventing premature PA channel formation—a process that inactivates the toxin. The role of ANTXR in PA oligomerization and in the stabilization of toxin complexes containing octameric PA are not understood.

Methodology

Using a fluorescence assembly assay, we show that the extracellular ANTXR domain drives PA oligomerization. Moreover, a dimeric ANTXR construct increases the extent of and accelerates the rate of PA assembly relative to a monomeric ANTXR construct. Mass spectrometry analysis shows that heptameric and octameric PA oligomers bind a full stoichiometric complement of ANTXR domains. Electron microscopy and circular dichroism studies reveal that the two different PA oligomers are equally stabilized by ANTXR interactions.

Conclusions

We propose that PA oligomerization is driven by dimeric ANTXR complexes on cell surfaces. Through their interaction with the ANTXR, toxin complexes containing heptameric and octameric PA oligomers are similarly stabilized. Considering both the relative instability of the PA heptamer and extracellular assembly pathway identified in plasma, we propose a means to regulate the development of toxin gradients around sites of infection during anthrax pathogenesis.     

Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient

Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (DeltapH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. DeltapH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or phi clamp) drives translocation synergistically under a DeltapH. We propose that a Brownian ratchet mechanism proposed earlier for the phi clamp is cooperatively linked to a protonation-state, DeltapH-driven ratchet acting trans to the phi-clamp site. In a sense, the channel functions as a proton/protein symporter.     

Assembly and disassembly kinetics of anthrax toxin complexes

Proteolytic activation of the protective antigen (PA) component of anthrax toxin allows it to self-associate into a ring-shaped homoheptamer, [PA(63)](7), which can bind the enzymatic components lethal factor (LF) and edema factor (EF). [PA(63)](7) is a pore-precursor (prepore), and under the low-pH conditions of the endosome, it forms a transmembrane pore that allows LF and EF to enter the cytosol. PA was labeled with donor and acceptor fluorescent dyes, and F?rster resonance energy transfer was used to measure the assembly and disassembly kinetics of the prepore complex in solution. The dissociation rate constant for [PA(63)](7) was 1 x 10(-)(6) s(-)(1) (t(1/2) approximately 7 days). In contrast, a ternary complex containing the PA-binding domain of LF (LF(N)) bound to a PA(63) dimer composed of two nonoligomerizing mutants dissociated rapidly (t(1/2) approximately 1 min). Thus, the substantial decrease in the rate of disassembly of [PA(63)](7) relative to the ternary complex is due to the cooperative interactions among neighboring subunits in the heptameric ring. Low concentrations of LF(N) promoted assembly of the prepore from proteolytically activated PA, whereas high concentrations inhibited assembly of both the prepore and the ternary complex. A self-assembly scheme of anthrax toxin complexes is proposed.     

Crystallographic studies of the anthrax lethal toxin

Anthrax lethal toxin comprises two proteins: protective antigen (PA; MW 83 kDa) and lethal factor (LF; MW 87 kDa). We have recently determined the crystal structure of the 735-residue PA in its monomeric and heptameric forms ( Petosa et al . 1997 ). It bears no resemblance to other bacterial toxins of known three-dimensional structure, and defines a new structural class which includes homologous toxins from other Gram-positive bacteria. We have proposed a model of membrane insertion in which the water-soluble heptamer undergoes a substantial pH-induced conformational change involving the creation of a 14-stranded β-barrel. Recent work by Collier's group ( Benson et al . 1998 ) lends strong support to our model of membrane insertion. 'Lethal factor' is the catalytic component of anthrax lethal toxin. It binds to the surface of the cell-bound PA heptamer and, following endocytosis and acidification of the endosome, translocates to the cytosol. We have made substantial progress towards an atomic resolution crystal structure of LF. Progress towards a structure of the 7:7 translocation complex between the PA heptamer and LF will also be discussed.     

Lethal factor of anthrax toxin binds monomeric form of protective antigen

Anthrax toxin consists of three components: the enzymatic moieties edema factor (EF) and the lethal factor (LF) and the receptor-binding moiety protective antigen (PA). These toxin components are released from Bacillus anthracis as unassociated proteins and form complexes on the surface of host cells after proteolytic processing of PA into PA20 and PA63. The sequential order of PA heptamerization and ligand binding, as well as the exact mechanism of anthrax toxin entry into cells, are still unclear. In the present study, we provide direct evidence that PA63 monomers are sufficient for binding to the full length LF or its LF-N domain, though with lower affinity with the latter. Therefore, PA oligomerization is not a necessary condition for LF/PA complex formation. In addition, we demonstrated that the PA20 directly interacts with the LF-N domain. Our data points to an alternative process of self-assembly of anthrax toxin on the surface of host cells.