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.     

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.     

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 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.     

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.     

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 toxins

Bacillus anthracis, the etiological agent of anthrax, secretes three polypeptides that assemble into toxic complexes on the cell surfaces of the host it infects. One of these polypeptides, protective antigen (PA), binds to the integrin-like domains of ubiquitously expressed membrane proteins of mammalian cells. PA is then cleaved by membrane endoproteases of the furin family. Cleaved PA molecules assemble into heptamers, which can then associate with the two other secreted polypeptides: edema factor (EF) and/or lethal factor (LF). The heptamers of PA are relocalized to lipid rafts where they are quickly endocytosed and routed to an acidic compartment. The low pH triggers a conformational change in the heptamers, resulting in the formation of cation-specific channels and the translocation of EF/LF. EF is a calcium- and calmodulin-dependent adenylate cyclase that dramatically raises the intracellular concentration of cyclic adenosine monophosphate (cAMP). LF is a zinc-dependent endoprotease that cleaves the amino terminus of mitogen-activated protein kinase kinases (Meks). Cleaved Meks cannot bind to their substrates and have reduced kinase activity, resulting in alterations of the signaling pathways they govern. The structures of PA, PA heptamer, EF, and LF have been solved and much is now known about the molecular details of the intoxication mechanism. The in vivo action of the toxins, on the other hand, is still poorly understood and hotly debated. A better understanding of the toxins will help in the design of much-needed anti-toxin drugs and the development of new toxin-based medical applications.Abbreviations CMG2 Capillary morphogenesis protein 2 - DTA Diphtheria toxin A chain - EF Edema factor - EFn N-terminal fragment of EF - ETx Edema toxin - GR Glucocorticoid receptors - GSK3 Glycogen synthase kinase 3 - I domain Integrin-like domain - iNOS Inducible nitric oxide synthase - LF Lethal factor - LFn N-terminal fragment of LF - LTx Lethal toxin - MAPK Mitogen-activated protein kinase - Mek MAPK kinases - PA Protective antigen - PA₂₀ 20-kDa N-terminal fragment of PA - PA₆₃ 63-kDa C-terminal fragment of PA - TEM8 Tumor endothelial marker 8     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Proton-coupled protein transport through the anthrax toxin channel

Anthrax toxin consists of three proteins (approx. 90kDa each): lethal factor (LF); oedema factor (OF); and protective antigen (PA). The former two are enzymes that act when they reach the cytosol of a targeted cell. To enter the cytosol, however, which they do after being endocytosed into an acidic vesicle compartment, they require the third component, PA. PA (or rather its proteolytically generated fragment PA63) forms at low pH a heptameric beta-barrel channel, (PA63)7, through which LF and OF are transported--a phenomenon we have demonstrated in planar phospholipid bilayers. It might appear that (PA63)7 simply forms a large hole through which LF and OF diffuse. However, LF and OF are folded proteins, much too large to fit through the approximately 15A diameter (PA63)7 beta-barrel. This paper discusses how the (PA63)7 channel both participates in the unfolding of LF and OF and functions in their translocation as a proton-protein symporter.     

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.     

Cys-Cys cross-linking shows contact between the N-terminus of lethal factor and Phe427 of the anthrax toxin pore

Electrophysiological studies of wild-type and mutated forms of anthrax protective antigen (PA) suggest that the Phe clamp, a structure formed by the Phe427 residues within the lumen of the oligomeric PA pore, binds the unstructured N-terminus of the lethal factor and the edema factor during initiation of translocation. We now show by electrophysiological measurements and gel shift assays that a single Cys introduced into the Phe clamp can form a disulfide bond with a Cys placed at the N-terminus of the isolated N-terminal domain of LF. These results demonstrate direct contact of these Cys residues, supporting a model in which the interaction of the unstructured N-terminus of the translocated moieties with the Phe clamp initiates N- to C-terminal threading of these moieties through the pore.     

PA63 channel of anthrax toxin: an extended beta-barrel

Anthrax toxin consists of three protein components: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA(63), generated by protease "nicking" of whole PA, is responsible for delivering the toxin's catalytic fragments (LF and EF) to the target cell's cytosol. In planar bilayer membranes, trypsin-nicked PA makes cation-selective voltage-gated channels with a pore diameter of > or =12 A. The channels are presumed to be heptameric "mushrooms", with an extracellular "cap" region and a membrane-inserted, beta-barrel "stem". Although the crystal structure of the water-soluble monomeric form has been resolved to 2.1 A and that of the heptameric "prepore" to 4.5 A, the structure for the membrane-bound channel (pore) has not been determined. We have engineered mutant channels that are cysteine-substituted in residues in the putative beta-barrel, and identified the residues lining the channel lumen by their accessibility to a water-soluble sulfhydryl-specific reagent. The reaction with lumen-exposed cysteinyl side chains causes a drop in channel conductance, which we used to map the residues that line the pore. Our results indicate that the beta-barrel structure extends beyond the bilayer and involves residues that are buried in the monomer. The implication is that major rearrangement of domains in the prepore cap region is required for membrane insertion of the beta-barrel stem.