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.     

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.     

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 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.     

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.     

Mechanism of membrane translocation by anthrax toxin: insertion and pore formation by protective antigen

Proteolytic activation of receptor-bound protective antigen (PA) at the cell surface removes PA₂₀, allowing PA₆₃ to oligomerize and form a ring-shaped heptameric prepore. The prepore binds edema factor (EF) and lethal factor (LF) and, after endocytosis and trafficking of the complex to an acidic, vesicular compartment, it undergoes membrane insertion and mediates translocation of EF/LF to the cytosol. Data from membrane conductance experiments support a model of membrane insertion in which the 2β2–2β3 loop of PA, which is disordered in native PA and the prepore, forms a 14-stranded transmembrane β-barrel. Recent studies on the process of prepore-to-pore conversion and our current understanding of the mechanism of pH-dependent translocation will be described.     

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.     

The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. Using single-channel electrophysiology, we show that PA channels contain two populations of conductance states, which correspond to two different PA pre-channel oligomers observed by electron microscopy—the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here, we report a 3.2-Å crystal structure of the PA octamer. The octamer comprises ∼ 20-30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus, the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity.     

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.     

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.     

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.     

Involvement of domain 3 in oligomerization by the protective antigen moiety of anthrax toxin

Protective antigen (PA), a component of anthrax toxin, binds receptors on mammalian cells and is activated by a cell surface protease. The resulting active fragment, PA(63), forms ring-shaped heptamers, binds the enzymic moieties of the toxin, and translocates them to the cytosol. Of the four crystallographic domains of PA, domain 1 has been implicated in binding the enzymic moieties; domain 2 is involved in membrane insertion and oligomerization; and domain 4 binds receptor. To determine the function of domain 3, we developed a screen that allowed us to isolate random mutations that cause defects in the activity of PA. We identified several mutations in domain 3 that affect monomer-monomer interactions in the PA(63) heptamer, indicating that this may be the primary function of this domain.     

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.     

Role of the Protective Antigen Octamer in the Molecular Mechanism of Anthrax Lethal Toxin Stabilization in Plasma

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-γ-D-glutamic acid capsule. Atx is comprised of three proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT can assemble on host cell surfaces or extracellularly in plasma. We show that, under physiological conditions in bovine plasma, LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel-forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration, which allows them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood.     

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.     

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.     

GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immunoelectron microscopy and gold-labelled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock-down of GRP78 results in the emergence of resistance to anthrax lethal toxin and oedema toxin action.     

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.     

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.     

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.