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 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.     

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.     

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.     

Interaction of the 20 kDa and 63 kDa fragments of anthrax protective antigen: kinetics and thermodynamics

The action of anthrax toxin begins when the protective antigen (PA(83), 83 kDa) moiety binds to a mammalian cell-surface receptor and is cleaved by a furin-family protease into two fragments: PA(20) (20 kDa) and PA(63) (63 kDa). After PA(20) dissociates, receptor-bound PA(63) spontaneously oligomerizes to form a heptameric species, which is able to bind the two enzymatic components of the toxin and transport them to the cytosol. Treatment of PA(83) with trypsin yielded PA(63) and a form of PA(20) lacking unstructured regions at the N- and C-termini. We labeled these fragments with dyes capable of fluorescence resonance energy transfer to quantify their association in solution. We kinetically determined that the equilibrium dissociation constant is 190 nM with a dissociation rate constant, k(off), of 3.3 x 10(-)(2) s(-)(1) (t(1/2) of 21 s). A two-step association process was observed using stopped-flow: a fast bimolecular step (k(on) = 1.4 x 10(5) M(-)(1) s(-)(1)) was followed by a slower unimolecular step (k = 3.5 x 10(-)(3) s(-)(1)) with an equilibrium isomerization constant, K(iso), of 2.1. The two-step mechanism most consistent with the data is one in which the dissociation of the PA(20).PA(63) complex is followed by an isomerization in the PA(63) moiety. Our results indicate that, following the cleavage of PA on the cell surface, PA(20) is largely dissociated within a minute. A slow isomerization step in PA(63) may then potentiate it for oligomerization and subsequent steps in toxin action.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

Fusions of anthrax toxin lethal factor to the ADP-ribosylation domain of Pseudomonas exotoxin A are potent cytotoxins which are translocated to the cytosol of mammalian cells.

The lethal factor (LF) and edema factor (EF) components of anthrax toxin are toxic to animal cells only if internalized by interaction with the protective antigen (PA) component. PA binds to a cell surface receptor and is proteolytically cleaved to expose a binding site for LF and EF. To study how LF and EF are internalized and trafficked within cells, LF was fused to the translocation and ADP-ribosylation domains (domains II and III, respectively) of Pseudomonas exotoxin A. LF fusion proteins containing Pseudomonas exotoxin A domains II and III were less toxic than those containing only domain III. Fusion proteins with a functional endoplasmic reticulum retention sequence, REDLK, at the carboxyl terminus of domain III were less toxic than those with a nonfunctional sequence, LDER. The most potent fusion protein, FP33, had an EC50 = 2 pM on Chinese hamster ovary cells, exceeding that of native Pseudomonas exotoxin A (EC50 = 420 pM). Toxicity of all the fusion proteins required the presence of PA and was blocked by monensin. These data suggest that LF and LF fusion proteins are efficiently translocated from acidified endosomes directly to the cytosol without trafficking through other organelles, as is required for Pseudomonas exotoxin A. This system provides a potential vehicle for importing diverse proteins into the cytosol of mammalian cells.     

Fragment complementation studies of protein stabilization by hydrophobic core residues

Interactions that stabilize the native state of a protein have been studied by measuring the affinity between subdomain fragments with and without site-specific residue substitutions. A calbindin D(9k) variant with a single CNBr cleavage site at position 43 between its two EF-hand subdomains was used as a starting point for the study. Into this variant were introduced 11 site-specific substitutions involving hydrophobic core residues at the interface between the two EF-hands. The mutants were cleaved with CNBr to produce wild-type and mutated single-EF-hand fragments: EF1 (residues 1--43) and EF2 (residues 44--75). The interaction between the two EF-hands was studied using surface plasmon resonance (SPR) technology, which follows the rates of association and dissociation of the complex. Wild-type EF1 was immobilized on a dextran matrix, and the wild-type and mutated versions of EF2 were injected at several different concentrations. In another set of experiments, wild-type EF2 was immobilized and wild-type or mutant EF1 was injected. Dissociation rate constants ranged between 1.1 x 10(-5) and 1.0 x 10(-2) s(-1) and the association rate constants between 2 x 10(5) and 4.0 x 10(6) M(-1) s(-1). The affinity between EF1 and EF2 was as high as 3.6 x 10(11) M(-1) when none of them was mutated. For the 11 hydrophobic core mutants, a strong correlation (r = 0.999) was found between the affinity of EF1 for EF2 and the stability toward denaturation of the corresponding intact protein. The observed correlation implies that the factors governing the stability of the intact protein also contribute to the affinity of the bimolecular EF1-EF2 complex. In addition, the data presented here show that interactions among hydrophobic core residues are major contributors both to the affinity between the two EF-hand subdomains and to the stability of the intact domain.     

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.     

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.