Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation

Abstract

The protective antigen (PA) moiety of anthrax toxin forms a heptameric pore in endosomal membranes of mammalian cells and translocates the enzymatic moieties of the toxin to the cytosol of these cells. Phenylalanine-427 (F427), a solvent-exposed residue in the lumen of the pore, was identified earlier as being crucial for the transport function of PA. The seven F427 residues were shown in electrophysiological studies to form a clamp that catalyzes protein translocation through the pore. Here, we demonstrate by a variety of tests that certain F427 mutations also profoundly inhibit the conformational transition of the heptameric PA prepore to the pore and thereby block pore formation in membranes. Lysine, arginine, aspartic acid, or glycine at position 427 strongly inhibited this acidic pH-induced conformational transition, whereas histidine, serine, and threonine had virtually no effect on this step, but inhibited translocation instead. Thus, it is possible to inhibit pore formation or translocation selectively, depending on the choice of the side chain at position 427; and the net inhibition of the PA transport function by any given F427 mutation is the product of its effects on both steps. Mutations inhibiting either or both steps elicited a strong dominant-negative phenotype. These findings demonstrate the dual functions of F427 and underline its central role in transporting the enzymatic moieties of anthrax toxin across membranes.

Toxins that modify targets within the cytosolic compartment of mammalian cells play crucial roles in many bacterial diseases. Most such toxins have separable catalytic and receptor-binding moieties; the receptor-binding moiety binds to cell surface receptors and facilitates delivery of one or more catalytic moieties to the cytosol (1). Besides binding to receptors, the receptor-binding moieties of some of these toxins, including anthrax toxin, also can insert into membranes and form ion-conductive pores (channels) that function in A-chain transport. The mechanisms of pore formation and membrane translocation by these toxins are of great interest in understanding how proteins translocate across membranes and for developing a firmer foundation for medical applications of toxins.

Anthrax toxin is a tripartite intracellularly acting toxin, composed of two catalytic moieties, edema factor (EF) and lethal factor (LF), and a single receptor-binding/pore-forming moiety, protective antigen (PA) (2). PA (83 kDa) binds to cell surface receptors and is cleaved by furin or a furin-like protease to an active, 63-kDa form (PA₆₃) (3). PA₆₃ oligomerizes into a heptameric, receptor-bound prepore, which contains high-affinity binding sites for EF and LF. The PA prepore can bind up to three molecules of EF and/or LF competitively (4–8), and the resulting toxin-receptor complexes are internalized by receptor-mediated endocytosis and trafficked to endosomes. There, the prepore undergoes an acidification-triggered conformational rearrangement to a cation-selective, transmembrane pore (9). Although the structure of PA pore has not been determined, there is strong evidence that during prepore–pore conversion the 2β2–2β3 loops (residues 302–325) in domain 2 of the prepore move to the base of the structure to form a 14-strand transmembrane β-barrel, similar to that formed by Staphylococcus aureus α-hemolysin (10–12) (Fig. 1). This transition is believed to yield a mushroom-shaped pore, which mediates translocation of EF and LF across the endosomal membrane. Within the cytosol, EF, an 89-kDa calmodulin-dependent adenylate cyclase, elevates the intracellular cAMP level (13), and LF, a 90-kDa zinc protease, inactivates mitogen-activated proteins kinase kinases (14).

Fig. 1.

Prepore-to-pore conversion and the location of F427 the prepore. (A) Upon acidification, the 2β2–2β3 loops in domain 2 move to the base of the heptamer to form a 14-strand transmembrane β-barrel. (Modified from ref. 12.) (B) Aerial view of the membrane-proximal face of PA heptameric prepore, with F427 residues modeled into the structure. (Modified from ref. 18.)

Membrane translocation is the least well understood aspect of the action of intracellularly acting toxins, and anthrax toxin has recently emerged as a tractable system for studying this process. Phenylalanine-427 (F427), a residue in the 2β10–2β11 loop on the luminal aspect of PA, is essential for the protein transport activity of the protein and has become a focal point in understanding the process of translocation. Attention was first drawn to F427 when the crystallographic structure of the prepore was solved, revealing this residue to be luminal and solvent-exposed (15), and thus a candidate for interacting with translocating polypeptides (Fig. 1). Mutation of F427 to Ala was found almost to abolish the ability of PA to mediate the entry of a model enzymatic A-chain into Chinese hamster ovary-K1 (CHO-K1) cells (16). Furthermore, F427A PA showed a dominant-negative phenotype, which depended on its ability to cooligomerize with the wild-type (WT) protein and form heptamers with attenuated transport activity (17). Subsequently, electrophysiological studies showed that pores formed by the F427A mutant in planar phospholipid bilayers were defective in catalyzing polypeptide translocation across these membranes (18). Evidence was presented that the seven F427 residues of the PA heptamer come into close proximity within the lumen of the pore and form a clamp, termed the Phe clamp, which catalyzes polypeptide translocation. The translocation defects caused by several F427 mutants in planar bilayers appeared to correlate approximately with the degree of impairment in mediating toxicity in CHO-K1 cells, suggesting that the biochemical defect might fully account for the loss of biological activity.

Here, we report that whereas certain F427 mutations primarily affect the translocation function of PA, others strongly inhibit the conformational transition of the prepore to the pore, and still others affect both steps. Thus, F427 participates in two sequential steps in the overall transport function of the toxin, and the net effect of any given mutation is the product of its effects on both steps. These findings have ramifications for understanding the mechanisms of pore formation, translocation, and the basis of the dominant-negative phenotype.

Results

Effects of F427 Mutations on the Ability of PA to Permeabilize Membranes to Monovalent Cations.

To investigate the role of F427 in PA pore formation, this residue was replaced with a series of other amino acids. The WT PA pore is cation-selective, and we tested the various PA F427 mutants for ability to form pores in liposomal membranes by means of a K⁺ release assay. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes containing KCl were added to acidic buffer (pH 5.0) followed by the addition of purified WT or mutant prepore complexed with the PA-binding domain of anthrax toxin receptor 2. K⁺ release was then monitored with a K⁺-selective electrode (Fig. 2A). The PA-binding domain of the receptor (R2) allowed the assay to be performed under conditions more closely resembling physiological (19).

Fig. 2.

Effects of F427 mutations on the ability of PA to permeabilize membranes to monovalent cations. (A) The K⁺ release assay was performed as described in Material and Methods. Representative recordings of K⁺ release from liposomes generated by the indicated PA prepores are shown. DNI and buffer were used as controls. (B) The rates of K⁺ release were calculated from three independent experiments shown in A and were expressed as fractions of that observed with WT PA. *, not detectable. (C) ⁸⁶Rb⁺ released from CHO-K1 cells by PA-WT and F427 mutants bound at the cell surfaces. Rb⁺ release by PA F427 mutants was expressed as fraction of the amount released with WT PA under the conditions of the assay.

Replacement of F427 with Arg, Lys, or Gly essentially ablated the ability of PA to release K⁺ from liposomes; release was comparable with that observed with buffer or with DNI, a double PA mutant (K397D, D425K) known to be strongly defective in pore formation (17). The rates of K⁺ release by F427S and F427H were similar to WT (90–110%), followed by Trp and Tyr (70%), Leu (50%), Ile and Ala (30%), and Asp (<10%) (Fig. 2B). Rates of release by the F427 mutants did not correlate with their unitary channel conductances measured in planar lipid bilayers (18). For example, the unitary conductance of F427A channel for K⁺ was 2-fold greater than that of WT, whereas the rate of K⁺ release from liposomes by this mutant was one-third that of WT. Thus, the effect on K⁺ release, at least for some mutations, could not be caused simply by an effect on unitary conductance, suggesting an inhibition of prepore–pore conversion.

Similar results were obtained when we tested the F427 mutants for ability to permeabilize receptor-bearing cellular membranes to the monovalent cation, ⁸⁶Rb⁺. We measured the activities of various F427 mutants in releasing ⁸⁶Rb⁺ from CHO-K1 cells preloaded with this isotope (Fig. 2C). CHO-K1 cells incubated overnight with ⁸⁶RbCl were washed, and WT or mutant prepore was allowed to bind to the cell surface in the cold. The cells were then washed, and ⁸⁶Rb⁺ release was measured after exposing the cells to low pH buffer. The pattern of release by the tested mutants closely resembled that observed in our measurements of K⁺ release from liposomes. The Gly, Arg, Lys, and Asp mutations strongly inhibited Rb⁺ release, whereas Ser, His, and hydrophobic residues, except for Ile, had little effect. The F427 mutations did not affect the ability of PA to bind to receptors (data not shown).

Effects of F427 Mutations on PA Insertion into Liposomal Membranes.

To monitor PA insertion into membranes directly, we labeled WT PA and F427 mutants with an environment-sensitive dye [N,N_-dimethyl-N(iodoacetyl)-N_-(7-nitrobenz-2-oxa-1,3-diazol)ethylenediamine (NBD)] attached to a Cys residue introduced at position 305. The side chain at position 305 projects from the β-barrel of the membrane-inserted pore into the lipid bilayer, and thus an increase in the intensity of NBD emission is seen upon membrane insertion as this group transitions from a polar to a nonpolar environment. The relative rates of membrane insertion by the various F427 mutants measured by this approach correlated with their activities in the K⁺-release and Rb⁺-release assays (Fig. 3).

Fig. 3.

Effects of F427 mutations on PA insertion into liposomal membranes as measured by NBD fluorescence. (A) Representative recordings of NBD emission at 544 nm are shown upon PA pore insertion as a function of time. (B) Initial rates of change of NBD emission intensity were calculated from three independent experiments, as shown in A, with the same double exponential equation used for analyzing the data from K⁺ release. The rates were expressed as fractions of PA-WT. *, not detectable.

Effects of F427 Mutations on the Conformational Transition of the Prepore to the Pore.

Coincident with the conformational transition of the prepore to the pore, triggered by acidic pH, the prepore is transformed into an SDS-resistant oligomeric form or forms (20–22). When this transition occurs in solution, these forms run on SDS/PAGE as a series of high-molecular mass bands, perhaps representing discrete aggregates. When the transition is triggered with prepore bound to cells, one observes a diffuse band at the same location in the gel. As shown in Fig. 4A, when acidified in solution, WT, F427W, F427L, F427I, F427Y, F427S, and F427H prepores converted to SDS-resistant forms efficiently, whereas F427A, F427R, and F427D showed reduced amounts of these forms. F427G gave a series of SDS-resistant bands that migrated somewhat faster than those formed by the other mutants or the WT. Similar results were observed when the WT and mutant prepores were bound to the plasma membrane of CHO-K1 cells before acidification. Little or no SDS-resistant material was formed with the Gly, Arg, and Asp mutants, and significant reductions could be seen with the Ile and Ala mutants (Fig. 4B). These findings support the hypothesis that some F427 mutations strongly affect the prepore-to-pore transition.

Fig. 4.

Effects of F427 mutations on the conformational transition of the prepore to the pore. (A) SDS-resistant PA₆₃ oligomers converted from the indicated PA prepores in acidified solution. Samples were separated by SDS/PAGE and visualized with Coomassie staining. (B) SDS-resistant PA₆₃ oligomers converted from the indicated PA prepores bound to the cell surfaces of CHO-K1 cells at pH 5.0. The samples were subjected to SDS/PAGE followed by Western blotting with a goat anti-PA antibody.

Correlation of the Effects of F427 Mutations on Toxicity with Effects on Pore Formation and Membrane Translocation.

The biological activity of WT PA and F427 mutants was determined by measuring the inhibition of protein synthesis in CHO-K1 cells in the presence of LF_N-DTA, the catalytic domain of diphtheria toxin (DTA) fused to the C terminus of LF_N (Table 1). DTA inhibits protein synthesis by catalyzing the inactivation of eukaryotic elongation factor 2 within the cytosol. Consistent with results reported earlier (18), the F427W, F427L, and F427Y mutants were highly active in mediating the cytotoxicity of LF_N-DTA and thus efficiently translocated LF_N-DTA across the endosomal membranes into the cytosol of CHO-K1 cells. The F427G, F427R, and F427D mutants were inactive, consistent with their severely impaired ability to form pores. However, despite having pore-forming activity equivalent to WT, the F427H, F427S, and F427T mutants were inactive in the cytotoxicity assay. To test the hypothesis that the His, Ser, and Thr mutations might cause strong defects in membrane translocation, we bound [³⁵S]methionine-labeled LF_N (³⁵S-LF_N) to WT or mutant PA at the surface of CHO-K1 cells and measured the level of ³⁵S-LF_N translocation across the plasma membrane after exposing the cells to acidic medium (Fig. 5). WT, F427W, F427L, and F427Y translocated ³⁵S-LF_N efficiently, whereas F427S, F427H, and F427T did not. Certain mutants, such as F427I and F427A, were inactive in the cytotoxicity assay by virtue of their partial impairment of both pore formation and translocation (Table 1).

Table 1.

Pore formation and cytotoxicity of WT PA and PA-F427X

PA	Pore formation†	Cytotoxicity‡
WT (F)	++++	+
F427W	++++	+
F427L	+++	+
F427I	++	−
F427Y	++++	+
F427A	++	−
F427G	−	−
F427S	++++	−
F427T	++++	−
F427H	++++	−
F427R	−	−
F427D	+	−
DNI	−	−

^†The pore formation activity of each protein was averaged from the rate of K⁺ release and initial rate of change of NBD fluorescence intensity. ++++, 75–100% activity of WT; +++, 50–75% of WT; ++, 30–50% of WT; +, 5–30% of WT; −, 0–5% of WT.

^‡+, cytotoxic; −, noncytotoxic. F427K had essentially the same phenotype as F427R.

Fig. 5.

Effects of F427 mutations on PA-mediated LF_N translocation across the plasma membrane. WT or F427 mutant prepore was bound to the surfaces of CHO-K1 cells in the cold followed by the addition of ³⁵S-LF_N to bind to the prepores on the cell surfaces. Translocation was induced by incubating the cells in pH 5 buffer at 37°C for 1–2 min. The cells were either directly lysed (pronase −) or treated with pronase (pronase +), followed by lysis. The cell lysates were subjected to SDS/PAGE followed by autoradiography. Pronase −, total ³⁵S-LF_N that was bound to the cell surfaces; Pronase +, ³⁵S-LF_N that was translocated across the plasma membrane.

A Strong Dominant-Negative Phenotype Can Be Generated by Mutations Affecting Pore Formation, Translocation, or Both.

The F427A mutant was shown earlier to cooligomerize with WT PA and exhibit a dominant-negative phenotype in blocking its transport activity (17). We hypothesized that some of the noncytotoxic F427 mutants could be dominant-negative inhibitors as the result of effects on either pore formation or translocation, or both. As shown in Fig. 6A, starting at molar ratio of 1:1, the F427D, F427R, F427H, and F427G mutants showed efficient inhibition of WT PA-mediated cytotoxicity with a potency that was similar to the dominant-negative inhibitor, DNI. To investigate at which steps the F427 mutants inhibited cytotoxicity, we monitored the SDS-resistant forms generated by WT PA and the mixture of F427X/WT (molar ratio, 1:1) as they were internalized into the cells (Fig. 6B). The F427G, F427R, and F427D mutants strongly inhibited the formation of SDS-resistant pores in the cells; F427A showed an intermediate level of inhibition; and F427I, F427S, and F427H caused no inhibition. Finally, a pronase protection assay was used to measure the translocation of LF_N across the plasma membrane in response to acidification of the external medium. Compared with WT PA, F427I partially inhibited LF_N translocation; and F427A, F427S, and F427H completely inhibited the translocation (Fig. 6C). Collectively, these results demonstrate that a strong dominant-negative phenotype can result from interference with pore formation, translocation, or both of these steps.

Fig. 6.

Dominant-negative effects of F427 mutants. (A) Dominant-negative effects of F427 mutants on cytotoxicity. The concentrations of PA₈₃-WT (0.2 nM) and LF_N-DTA (1 nM) were kept constant. The molar ratios of F427X/WT ranged from 1/8 to 4 as indicated. The levels of protein synthesis were measured by detecting the [³H]leucine incorporation and expressed as fractions of PA-WT. (B) Dominant-negative effects of F427 mutants on pore formation. PA₈₃-WT or the mixture of PA₈₃-F427X and PA₈₃-WT (molar ratio of F427X to WT, 1:1) was incubated with CHO-K1 cells at 37°C for 1 h. The SDS-resistant PA₆₃ oligomers converted from the PA internalized into endosomes were detected by Western blotting as described in Fig. 4B. (C) Dominant-negative effects of F427 mutants on LF_N translocation across the cell membrane. PA₈₃-WT or the mixture of PA₈₃-F427X and PA₈₃-WT (F427X/WT = 1:1) was prebound to the surfaces of CHO-K1 cells in the cold followed by the addition of ³⁵S-LF_N to the cells. Translocation was triggered by incubating the cells in acidic buffer. The cells were either directly lysed (Pronase −) or treated with pronase (Pronase +) followed by lysis. ³⁵S-LF_N was detected by autoradiography.

Discussion

Recent advances in understanding the structure and activity of the pore formed by PA have made anthrax toxin attractive for investigating how large globular proteins can be transported through a proteinaceous pore. In this work, we describe mutations at a single residue, F427, that differentially affect two sequential steps in protein transport: pore formation and translocation through the pore.

After the discovery that the F427A mutation strongly inhibited the transport of intracellular effectors into CHO-K1 cells (16), studies in planar phospholipid bilayers provided a possible explanation of how this mutation disrupted PA biological function (18). F427A and certain other F427 mutations were shown to inhibit ΔpH-driven (or Δψ-driven) translocation of LF_N through PA pores in these model membranes (23). Furthermore, F427 was shown to form a seal around the translocating polypeptide chain against the passage of ions, a seal perhaps analogous to that formed by aliphatic side chains at a constriction point in the pore formed in the Sec61 protein secretion channel (24). Consistent with this model, site-directed spin-labeling measurements indicated that the F427 side chains, which are ≈15–20 Å apart in the prepore, were drawn into close proximity (≤10 Å) during the conformational rearrangement of the prepore to the pore (18). These findings and others led to the proposal that the seven F427 residues formed a structure, dubbed the Phe clamp, at a constriction point in the translocation pathway and that this structure catalyzes transport of polypeptides through the pore by means of a charge-state dependent, ΔpH-driven Brownian ratchet mechanism.

Against this backdrop, we report that replacing F427 with certain amino acids has a profound inhibitory effect on pore formation per se, by blocking the conformational rearrangement of the prepore to the pore. The net effect of any mutation at this site on toxicity therefore involves effects on both the pore formation and the translocation steps in transporting substrate proteins across endosomal membranes. We found that replacement of F427 with Gly or a charged residue, such as Arg, strongly impaired prepore-to-pore conversion, and several other mutations moderately inhibited this step. Prepore-to-pore conversion and translocation showed similarities in side-chain dependence at position 427, in that hydrophobic residues, except Ile, were active in promoting both of these steps, and Gly or residues with a charged side chain blocked both steps. However, F427S, F427T, and F427H did not impair prepore-to-pore conversion but strongly inhibited translocation. Thus, at least for some mutations at this site, different mechanistic explanations must be invoked to explain the differential effects of F427 mutations on pore formation and translocation.

How might a F427 mutation inhibit prepore-to-pore conversion? A plausible mechanism is suggested by electron paramagnetic resonance measurements indicating that the F427 side chains are close to one another (≤10 Å) in the pore. Depending on the closeness of approach of side chains at this position within the pore, or in intermediate states in the process of prepore-to-pore conversion, hydrophobic and/or stacking interactions between aromatic (Trp, Tyr, and His) or aliphatic side chains (Leu), or hydrogen bonds between certain others (e.g., Ser, Thr, or His), could foster prepore-to-pore conversion, whereas electrostatic repulsion between charged side chains, e.g., Arg, could inhibit this conversion. The slightly weaker inhibition observed with F427D, relative to F427R and F427K, could reflect the high density of acidic residues in the pore lumen, implying a mildly acidic local pH, and thus partial protonation of an acidic residue at position 427. Consistent with this notion and the fact that Glu has a higher pK_a than Asp, the F427E mutation caused less inhibition of K⁺ release than the F427D mutation (data not shown). The basis of the strong inhibitory effect of the F427G mutation is unclear, but the consistently lower activity of Ile (β-branched) relative to Leu (γ-branched) illustrates the exquisite sensitivity of the prepore-to-pore transition to side-chain geometry at position 427.

The selective inhibition of the translocation step by certain mutations, notably F427S, F427T, and F427H, could have various explanations. Whereas WT PA was found in single-channel (single-pore) conductance measurements to interact with LF_N in such a manner as to form a seal against ion passage, the F427A pores gave an incomplete seal and a flickering conductance pattern (18). An incomplete seal could disrupt the transmembrane gradient and hence the driving force for translocation. A similar mechanism may underlie the effects of the Ser and Thr mutations on translocation, because of their relatively small side-chain volumes. However the inhibitory effect of the F427H mutant may depend on its ability to be protonated, inasmuch as a positive charge at this position would be predicted to inhibit interactions with the positively charged N-terminal translocation leader sequences of LF and EF. It is also possible that the capacity of His, Ser, and Thr to form hydrogen bonds, between each other or with translocating polypeptides, may be related to their blockage of translocation.

The F427D, F427R, F427H, and F427G mutants exhibited a strong dominant-negative phenotype, with inhibitory potency similar to that of DNI. F427D, F427R, and F427G inhibited pore formation, whereas F427S and F427H specifically inhibited membrane translocation. This finding indicates that major blockage of either prepore-to-pore conversion or translocation can yield such a phenotype. Both types of the mutants therefore cooligomerize with WT to yield inactive complexes. Dominant-negative forms of PA have been of interest both as potential inhibitors of anthrax toxin and as components of conjugate vaccines containing capsular material from Bacillus anthracis covalently linked to PA (25). The results presented here cast a new perspective on these applications.

Materials and Methods

Cell Culture and Media.

CHO-K1 cells were from the American Type Culture Collection and were grown in Ham's F-12 medium supplemented with 10% calf serum, 2 mM glutamine, 500 units/ml penicillin, and 500 units/ml streptomycin sulfate, under a humidified atmosphere with 5% CO₂.

Site-Directed Mutagenesis and Protein Purification.

Mutations at F427 were generated by site-directed mutagenesis (QuikChange; Stratagene) and were confirmed by DNA sequencing. Recombinant PA, R2, His-R2, and LF_N-DTA were expressed and purified as described in ref. 19. R2 LF_N-DTA and prepore were prepared as described in ref. 26. Protein concentrations were determined by spectrophotometry (A₂₈₀) and densitometry on SDS/PAGE with BSA as standard.

Liposome Preparation.

Liposomes were prepared as described in ref. 19. Briefly, DOPC, either alone or mixed with a Ni²⁺-chelating lipids, DOGs-NTA-Ni (1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl nickel salt}) (molar ratio, 100:8), in chloroform (Avanti Polar Lipids) was dried under N₂ gas to form a lipid film, followed by vacuum for 3 h to remove residual solvent. The dried lipid film was rehydrated with buffer to form multilamellar vesicles and subjected to three freeze-thaw cycles and extrusion through a 200-nm pore size polycarbonate filter (Nucleopore) in a mini extruder (Avanti Polar Lipids). The protocol yielded large unilamellar vesicles with an average diameter of 150–200 nm.

K⁺ Release Assay.

Liposomes containing 150 mM KCl, 10 mM Hepes (pH 7.4) were transferred into 150 mM NaCl, 20 mM Tris·HCl (pH 8.5) by buffer exchange, as described in ref. 19. The liposomes were added to K⁺ release buffer (50 mM sodium acetate, 150 mM NaCl, pH 5.0) and after 1 min, PA prepore (3 nM) complexed with R2 (40 nM) was added. The solution was stirred continuously with a magnetic stirrer, and K⁺ release was monitored with a K⁺-selective electrode (Orion Research). Rates of K⁺ release were calculated in SigmaPlot by fitting the K⁺ release curves to the double exponential equation f = a*[1 − exp(−b*x)] + c*[1 − exp(−d*x)], in which b is the rate constant of K⁺ release by PA and d is the rate constant of spontaneous K⁺ leakage from liposomes.

Time-Lapse Intensity Measurements of NBD Emission.

PA proteins carrying the mutation G305C were labeled with NBD as described (19, 26). The labeling efficiency was 90–100%. NBD emission of PA pores in the liposomal membranes was measured as described in ref. 19. Briefly, the liposomes were incubated with trypsin-activated PA (0.5 μM) and His-R2 (1 μM) at pH 8.5 for 30 min, and the protein-liposome mixtures were transferred to a cuvette with a stirring bar in an ISS K2 fluorometer. NBD was excited at 488 nm, and emission was recorded at 544 nm. Crossed polarizers on excitation, emission beams, and a 520-nm filter were used to reduce the background scatter. After addition of 1/10 volume of 1 M sodium acetate (pH 5.0) to the cuvette, the shift of NBD label from a polar (solution) to a nonpolar environment (liposomal membrane) was monitored. Rates of NBD emission were calculated in SigmaPlot by fitting the curves to the single exponential equation f = y0+a*[1 − exp(−b*x)], in which b is the rate of NBD emission at 544 nm.

Rb⁺ Release.

As described in ref. 27, CHO-K1 cells were incubated overnight in complete F-12 medium containing 1 μCi/ml ⁸⁶Rb⁺. After removal of the excess ⁸⁶Rb⁺, the cells were incubated with PA prepore (1 μg/ml) in F-12 medium at 4°C for 1 h. Unbound protein was removed by washing with cold PBS, and the cells were incubated with pH 5.0 or pH 8.0 buffer (140 mM NaCl, 20 mM Mes, 5 mM gluconic acid, pH 5.0 or pH 8.0) at 37°C for 5 min and subsequently incubated in 4°C for 30 min. Supernatants containing the released ⁸⁶Rb⁺ were collected and counted in a gamma-counter.

SDS-Resistant PA₆₃ Oligomer.

To assay prepore samples in solution, prepore (3 mg/ml) was incubated with R2 (2 mg/ml) in 20 mM Tris·HCl, 150 mM NaCl (pH 8.5) at room temperature for 30 min. The solution was acidified by the addition of 1/10 volume of 1 M sodium acetate (pH 5.0) and incubated for 10 min. Samples were exposed to 1.25% SDS for 20 min and electrophoresed on SDS/PAGE, and the bands were visualized after Coomassie staining. To assay prepore samples bound to the plasma membrane, CHO-K1 cells were incubated with PA prepores (10 μg/ml) for 1 h at 4°C. The cells were washed with cold PBS and incubated in 150 mM NaCl, 20 mM Mes, 5 mM gluconic acid, pH 5.0 buffer for 10 min at 4°C. The cells were then harvested and exposed to 1.25% SDS at 100°C for 10 min. The samples were subjected to SDS/PAGE followed by Western blotting with a goat anti-PA antibody (List Biolab), mouse anti-goat horseradish peroxidase (Santa Cruz), and SuperSignal Western detection reagent (Pierce). For assays of samples internalized to the endosome, cells were incubated with WT monomeric PA alone or mixed in a 1:1 ratio with mutant PA (10 μg/ml) for 1 h at 37°C. The cells were washed, harvested, and exposed to 1.25% SDS with 10-min boiling. The SDS/PAGE and detection of PA were performed as described above.

Dominant-Negative Inhibition of Anthrax Toxin-Mediated Cytotoxicity.

As described (28, 29), a mixture of various amounts of PA₈₃-F427 mutant with a constant amount of PA₈₃-WT (0.2 nM) and LF_N-DTA (1 nM) was added to CHO-K1 cells and incubated at 37°C for 4 h. The medium was removed and replaced with leucine-free F-12 medium supplemented with 1 μCi/ml [³H]leucine (NEN) and incubated at 37°C for 1 h. The inhibition of PA-dependent cell killing was determined by measuring [³H]leucine incorporated into cellular protein.

PA-Mediated LF_N Translocation Across the Plasma Membrane.

As described in ref. 30, CHO-K1 cells were incubated with PA (10 μg/ml) for 2 h at 4°C. Cells were then washed with cold PBS to remove unbound protein, and ³⁵S-LF_N, produced from TNT coupled reticulocyte lysate system (Promega), was added. The cells were then incubated for 2 h at 4°C. The unbound ³⁵S-LF_N was removed by washing, and ³⁵S-LF_N translocation was triggered by acidification of the cells with pH 5.0 buffer [150 mM NaCl, 20 mM Mes, 5 mM gluconic acid (pH 5.0)] at 37°C for 1–2 min. Pronase (2 mg/ml) was added to remove LF_N remaining on the cell surface after translocation. The cells were harvested, and the lysates were applied to SDS/PAGE followed by autoradiography.