From the Department of Microbiology and Molecular
Genetics, Harvard Medical School, Boston, Massachusetts 02115 and the
§ Department of Neuroscience, Albert Einstein College of
Medicine, Bronx, New York 10461
Received for publication, September 11, 2000, and in revised form, November 14, 2000
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ABSTRACT |
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The protective antigen (PA) moiety of anthrax
toxin delivers the toxin's enzymatic moieties to the cytosol of
mammalian cells by a mechanism associated with its ability to
heptamerize and form a transmembrane pore. Here we report that
mutations in Lys-397, Asp-425, or Phe-427 ablate killing of
CHO-K1 cells by a cytotoxic PA ligand. These mutations blocked PA's
ability to mediate pore formation and translocation in cells but had no
effect on its receptor binding, proteolytic activation, or ability to
oligomerize and bind the toxin's enzymatic moieties. The
mutation-sensitive residues lie in the
2 The mechanisms by which intracellularly acting toxins cross
membranes to access their cytosolic substrates are poorly understood. Most members of this class of toxins are bipartite entities (so-called AB toxins), composed of an enzymatic A polypeptide covalently or
noncovalently linked to a B polypeptide or complex (1). The B moiety
binds to cell-surface receptors and in general serves as a vehicle to
deliver the A moiety to the cytosol. There, the latter covalently
modifies its target substrate(s), eliciting disease symptoms.
Anthrax toxin (ATx)1 belongs
to a unique subset of AB toxins, termed binary toxins, in which the A
and B moieties are released from the bacteria as discrete monomeric
proteins. These proteins assemble at the surface of mammalian cells
into receptor-bound toxic complexes. The anthrax bacillus secretes two
different A proteins, edema factor (EF) and lethal factor (LF), plus a
single B protein, protective antigen (PA), which delivers them both to the cytosol. EF is an adenylate cyclase, whereas LF is a
Zn2+-dependent protease, which cleaves certain
mitogen-activated protein kinase kinases within mammalian cells
(2-4).
In some AB toxins, including ATx, diphtheria toxin, and several
clostridial toxins, the B moiety is capable of forming channels (pores)
in lipid bilayers under conditions that promote translocation (5-9).
Evidence from mutational studies with anthrax and diphtheria toxins
implies a close relationship between channel formation and the process
by which the A moieties cross cellular membranes (10, 11).
PA, a monomeric 83-kDa protein, binds to an as yet unidentified
cell-surface receptor on mammalian cells and is activated by furin or a
furin-like protease (12, 13). The resulting N-terminal fragment
(PA20; 20 kDa) diffuses into the extracellular milieu,
leaving the complementary C-terminal fragment (PA63; 63 kDa) bound to the receptor. PA63 manifests a set of
properties, not found in the native protein. PA63
spontaneously oligomerizes to form a ring-shaped heptamer,
(PA63)7 (14, 15), and binds EF and LF tightly
and competitively (16, 17). According to our current model,
(PA63)7 complexed with EF and/or LF is taken into the cell by receptor-mediated endocytosis and trafficked to an
acidic compartment (18). There, acidification induces a conformational
change in the (PA63)7 moiety, causing it to
convert to a membrane-spanning pore (19). Pore formation triggers
translocation of EF and/or LF across the membrane to the cytosol, but
the mechanism of this process remains to be elucidated.
The crystallographic structure of PA reveals a long, flat molecule,
consisting largely of 7-2
8 and
2
10-2
11 loops of domain 2 and are distant
both in primary structure and topography from the 2
2-2
3 loop, which is believed to
participate in formation of a transmembrane
-barrel. These results
suggest that Lys-397, Asp-425, and Phe-427 participate in
conformational rearrangements of a heptameric pore precursor that are
necessary for pore formation and translocation. Identification of these
residues will aid in elucidating the mechanism of translocation and may
be useful in developing therapeutic and prophylactic agents against anthrax.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-structure, and is organized into four
structural domains (Fig. 1A)
(15). Domain 1 contains the furin site, and the surface of this domain
exposed when PA63 is removed has been hypothesized to serve
as the binding site for EF and LF (15). Domain 2 forms the central
structure of the heptamer and contains a disordered loop
(2
2-2
3, residues 302-325) that has been
implicated in forming the transmembrane pore. Mutations have been found
recently within domain 3 that affect oligomerization, suggesting this
to be the function of this
domain.2 Domain 4 functions
in receptor binding, as demonstrated by mutational analysis and by
studies with monoclonal antibodies (11, 20).
View larger version (40K):
[in a new window]
Fig. 1.
Ribbon diagrams of the 63-kDa form of
PA. Domain 1' is shown in yellow, domain 2 in red, domain 3 is in
blue, and domain 4 is in green. The locations of Lys-397,
Asp-425, and Phe-427 are shown in a ribbon diagram of a single monomer
(A) from the PA63 heptamer. The dashed
line represents the disordered 2 2-2
3
loop that is believed to form a transmembrane
-barrel upon pore
formation. The crystallographic PA63 heptamer
(B) is also illustrated to show the approximate locations of
these residues in that structure (only Lys-397 is indicated, for
simplicity). This figure was prepared with Molscript (34).
The crystallographic structure of a heptameric form of PA63 has also been solved (Fig. 1B) (15). The structure shows a hollow ring, 160 Å in diameter and 85 Å high, with a central negatively charged lumen of average diameter 35 Å. There are no major conformational differences between PA63 in (PA63)7 and in native PA. The monomers pack such that domain 1' (that portion remaining after removal of PA20) and domain 2 face the lumen, whereas domains 3 and 4 are on the periphery. No hydrophobic surface that could interface with the core of a bilayer is seen, leading to the suggestion that this form may represent an intermediate in pore formation assembled on the cell surface.
Channel conductance experiments indicate that the
22-2
3 loops of the seven monomers of
heptameric PA63 interact to generate a transmembrane
-barrel similar to that observed in Staphylococcus aureus
-hemolysin (21, 22). These loops project outwards laterally from the
midsection of the crystallographic (PA63)7 and
would have to move to the base of this structure to assemble into a
-barrel on the heptamer axis. This movement could be effected by an
unfolding of the Greek-key motif comprising the first four
-strands
of domain 2, in which the 2
2 and 2
3
strands flanking the loop peel away from the edge of domain 2 (15).
In the current study, we show that mutation of any of three
solvent-exposed residues within domain 2, Lys-397, Asp-425, and Phe-427, blocks pore formation and translocation across native membranes. These residues are in the
27-2
8 and
2
10-2
11 loops, which lie on the luminal
aspect of the crystallographic heptamer, and are far removed from the
2
2-2
3 loop implicated in formation of the
transmembrane
-barrel. The characteristics of these mutant proteins
suggest that Lys-397, Asp-425, and Phe-427 participate in
conformational rearrangements of the PA63 heptamer that are required for translocation.
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EXPERIMENTAL PROCEDURES |
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Cell Culture, Media, and Chemicals-- Chinese hamster ovary-K1 (CHO-K1) cells were obtained from American Type Culture Collection. The cells were grown in Ham's F-12 medium supplemented with 10% calf serum, 2 mM L-glutamine, 500 units/ml penicillin G, and 500 units/ml streptomycin sulfate and maintained at 5% CO2 in a humidified atmosphere. Cells were seeded into 24- or 96-well microtiter plates (Costar, Cambridge, MA) 16-18 h prior to the experiment. All supplies for cell culture media were obtained from Life Technologies, Inc. unless noted otherwise. All chemicals were obtained from Sigma Chemical Co. unless specified.
Preparation of PA Proteins--
Mutations were constructed using
the QuikChange method of site-directed mutagenesis following the
manufacturer's protocol (Stratagene, La Jolla, CA). All proteins were
cloned into the pET22-b(+) (Novagen, Madison, WI) expression vector,
transformed into Escherichia coli BL21(DE3) (Novagen), and
expressed as described previously (23). Briefly, cultures were grown in
LB (24) at 37 °C to A600 of 1.0. Expression
of the recombinant protein was induced by the addition of
isopropyl--D-thiogalactopyranoside to 1 mM. Following induction, the cells were grown for an
additional 3 h at 30 °C and harvested by centrifugation for 10 min at 8000 × g.
Proteins were released from the periplasm by osmotic shock. The cells
were resuspended in 20 mM Tris, pH 8.0, 30% glucose, and 1 mM EDTA and incubated at room temperature for 10 min with continuous stirring. The cells were again harvested by centrifugation, resuspended in ice-cold 5 mM MgSO4 containing
20 mM benzamidine (Research Organics, Cleveland, OH), and
incubated at 4 °C for 10 min with constant stirring. After the cells
were again pelleted by centrifugation at 8000 × g, the
periplasmic extract was decanted. Tris-HCl, pH 8.0, was added to the
extract to a final concentration of 20 mM, and the entire
sample was loaded onto a column packed with Q-Sepharose High
Performance (Amersham Pharmacia Biotech, Piscataway, NJ). After the
column was washed with buffer A (20 mM Tris, pH 8.0), bound
proteins were eluted with a linear gradient of 0-25% buffer B (20 mM Tris, pH 8.0, 1 M NaCl). The PA-containing fractions were concentrated, and the buffer was exchanged over a pd-10
column (Amersham Pharmacia Biotech) equilibrated in buffer A. The
PA-containing eluate was loaded onto a Mono-Q (Amersham Pharmacia
Biotech) column and eluted with a 0-25% B gradient. Fractions
containing PA were determined by SDS-PAGE and stored at 80 °C.
Proteins were assayed using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) as per the manufacturer's instructions. All liquid chromatography was done using an AKTA purifier (Amersham Pharmacia Biotech).
Activation of PA-- PA was activated by treatment with trypsin, yielding nicked PA (nPA). Trypsin was added to PA (0.2-0.5 mg/ml) at a final trypsin:PA ratio of 1:1000 (w/w). This mixture was incubated at room temperature for 20 min followed by addition of a 10-molar excess of soybean trypsin inhibitor.
Cell Surface Translocation Assay--
A cell surface
translocation assay to measure the PA-mediated translocation of
[35S] LFN was performed as described (25).
CHO-K1 cells (2 × 105 cells/well) in a 24-well
plate were chilled on ice for 30 min. Medium was removed by aspiration,
and the cells were washed with ice-cold sterile Dulbecco's PBS. nPA
(2 × 10
8 M) in 250 µl of Ham's F-12
medium buffered with 10 mM HEPES, pH 7.4, was layered on
the cells and incubated for 2 h on ice. The medium was removed by
aspiration, and the cells were washed with sterile ice-cold Dulbecco's
PBS. The cells were then incubated on ice for 1 h with Ham's F-12
medium buffered with 10 mM HEPES, pH 7.4, containing
[35S] LFN produced by in vitro
transcription/translation using the TnT Coupled Reticulocyte Lysate
System (Promega, Madison, WI). After the removal of the
[35S] LFN, the cells were washed with
ice-cold PBS followed by a pH 5.0 or 7.0 pulse (10 mM Tris,
5 mM sodium gluconate, 140 mM NaCl, pH 5.0 or
7.0) at 37 °C for 1 min. The cells were then treated with 2 mg/ml
Pronase in 10 mM HEPES, pH 7.4, for 7 min at 37 °C. Pronase was omitted from some wells, as a cell-binding control. Cells
were lysed in 100 µl of lysis buffer (20 mM sodium
phosphate, pH 7.4, 10 mM EDTA, 1% Triton X-100) at 4 °C
for 10 min. Following lysis, the [35S] LFN in
the supernatant was counted in a scintillation counter. The percentage
of translocation was defined as: dpm protected from Pronase/dpm bound
to cells × 100.
Inhibition of Protein Synthesis--
Inhibition of protein
synthesis by LFNDTA was used to measure the overall ability
of PA to deliver a ligand to the cytosol (26). CHO-K1 cells were plated
at 2.5 × 104 cells/well in a 96-well plate 16 h
prior to the addition of protein. PA (1 × 1012 to
1 × 10
7 M) was incubated with cells in
the presence of 1 × 10
8 M
LFNDTA for 4 h at 37 °C. The medium was then
removed and replaced with leucine-free Ham's F-12 medium supplemented
with 10% calf serum, 2 mM L-glutamine, 500 units/ml penicillin G, 500 units/ml streptomycin sulfate, and
[3H]Leu at 1 µCi/ml. After incubation for 1 h at
37 °C, the cells were washed with ice-cold PBS followed by ice-cold
10% trichloroacetic acid. Protein synthesis was measured as the
quantity of [3H]Leu incorporated into the trichloroacetic
acid-precipitable material.
Heptamer Formation-- Oligomerization of PA63 was measured by incubating nPA (0.5 mg/ml at pH 8.0) with an equimolar amount of LFN for 30 min at room temperature. The samples were then subjected to electrophoresis in a 4-12% native gradient gel (FMC Bioproducts, Rockland, ME) using 50 mM CHES, pH 9.0, 2 mg/ml CHAPS as the running buffer. SDS-resistant heptamer was formed by adding 100 mM sodium acetate, pH 4.0, until the pH of the solution reached 5.0, after which the samples were incubated at room temperature for 30 min. The samples were then dissolved in SDS-PAGE sample buffer (24) and run on a 4-12% SDS-PAGE gradient gel (FMC Bioproducts). Proteins were visualized with Coomassie Brilliant Blue.
Rubidium Release--
CHO-K1 cells were plated at a density of
2 × 105 cells/well in 24-well plates and incubated at
37 °C for 24 h. The medium was aspirated and replaced with
medium containing 1 µCi/ml 86Rb and incubated for 16 h. Cells were then chilled on ice for 20 min followed by the removal of
the medium, washing of the cells, and addition of nPA (2 × 108 M) in HEPES-buffered medium. The cells
were incubated with nPA for 2 h on ice followed by the addition of
ice-cold pH 5.0 buffer. After a 30-min incubation, samples of the
supernatant were collected and the radioactivity was measured.
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RESULTS |
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Mutations at Lys-397 and Asp-425 Block PA-dependent
Cell Killing--
In exploring various regions of PA, we mutated
residues within the 27-2
8 and
2
10-2
11 loops, which lie on the luminal
aspect of the heptamer structure. Early in these studies, we found a residue in each loop that was crucial to PA's ability to deliver a
toxic ligand to the cytosol (23, 26). The ligand employed, LFNDTA, is a fusion protein containing LFN, the
PA-binding domain of LF, fused to DTA, the enzymic domain of diphtheria
toxin. Delivery of the fusion protein to the cytosol allows the DTA
moiety to catalyze the ADP-ribosylation of elongation factor-2, thereby inhibiting protein synthesis. As shown in Fig.
2, substitution of Ala for either Lys-397
(within the 2
7-2
8 loop) or Asp-425 (within 2
10-2
11) completely blocked PA's
ability to mediate LFNDTA-dependent inhibition
of [3H]Leu incorporation in CHO-K1 cells.
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The Mutations Specifically Block Translocation and Pore
Formation--
To identify the step in toxin action affected by these
mutations, we first examined the effects of K397A and D425A on
translocation, using the cell-surface assay of Wesche et al.
(25). Radiolabeled LFN was bound to trypsin-nicked PA (nPA)
at the cell surface, and the cells were briefly treated with low pH
buffer to induce translocation across the plasma membrane. The cells
were then treated with Pronase to digest any LFN remaining
exposed, and the translocated LFN was quantified by
scintillation counting. Both K397A and D425A inhibited translocation at
least 10-fold, as shown in Fig.
3B.
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The mutations did not diminish the amount of LFN initially
bound by PA63 at the cell surface (Fig. 3A) and
thus did not alter PA's receptor-binding and ligand-binding functions.
Furthermore, the specificities of cleavage at the furin site of PA by
trypsin or in the 22-2
3 loop by
chymotrypsin are retained in the mutant PAs (data not shown) (23).
These findings are strong indications that the K397A and D425A
mutations did not cause general misfolding of the protein.
The ability of PA to translocate ligands across membranes has been
correlated with its activity in forming transmembrane pores (11, 23).
We measured the pH-dependent PA-mediated release of
86Rb from 86Rb-preloaded CHO-K1 cells as an
assay of permeabilization of the plasma membrane (27). K397A and D425A
entirely abrogated pore formation as assessed by this parameter (Fig.
4). Pore formation in a planar lipid
bilayer is a highly sensitive method to assay for channel formation by
PA (6), and the mutations also blocked pore formation by
trypsin-activated PA in this system (data not shown). Thus the
inhibition of translocation by K397A and D425A is correlated with a
lesion in PA's ability to form pores.
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Oligomerization Is Not Affected by the Mutations but Transition to
an SDS-resistant Form Is--
To identify the step in pore formation
affected by the mutations, we first probed the ability of the mutant
forms of PA63 to oligomerize (15, 23). Addition of an
equimolar amount of LFN to nPA in solution promotes
oligomerization by PA63, and the resulting
LFN·(PA63)7 complex
migrates as a low mobility band in nondenaturing polyacrylamide gels.
As shown in Fig. 5, wild-type PA,
PA-K397A, and PA-D425A formed similar amounts of the LFN
complex, implying that oligomerization of PA63 is not
affected by the mutations.
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This conclusion is supported by results of anion-exchange (MonoQ) chromatography of the nicked mutant and wild-type PAs. PA63 elutes from the MonoQ resin in a single peak corresponding to an oligomeric form (or forms). Recently, PA mutants have been identified that are deficient in oligomerization, and such mutants yield little or no material in this peak.3 In contrast, K397A- and D425A-PA63 gave elution profiles closely resembling that of wild-type PA. The presence of (PA63)7 in the eluate was confirmed by electron microscopy (data not shown).
If maintained in buffer of pH 8.0, oligomeric PA63
dissociates in the presence of SDS into monomeric subunits. When
exposed to pH
7, or when treated with
-octylglucoside (even
at pH
8.0), the material converts to an SDS-resistant state and
runs as a high molecular mass band on SDS-PAGE (14, 22, 23). Unlike
wild-type preparations, LFN-liganded K397A or D425A prepore failed to convert to SDS resistance when treated at pH 5 (Fig. 6) or when incubated with
-octylglucoside (data not shown). Also, no SDS-resistant oligomer
was formed when cells containing bound mutant protein were treated at
low pH. These findings suggest that the K397A and D425A mutations
affect a conformational transition in pore formation.
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Effects of Other Mutations-- We examined the effects of substituting other amino acids besides Ala at positions 397 and 425 (Table I). Although K397D gave the same phenotype as K397A, K397R caused little loss of activity, indicating that a positive charge is important for function at this site. The effects of K397Q coincided with those of K397D and K397A, except that K397Q did not block conversion of the prepore to an SDS-resistant state. At position 425, all other substitutions tested, including the most conservative ones (Glu and Asn), had the same effect as the Ala substitution. Thus the requirement for Asp at this position is highly specific.
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We also mutated residues flanking Lys-397 in the
27-2
8 loop and Asp-425 in
2
10-2
11. Among the mutations examined
(see Table I) only F427A caused a major functional defect. Like K397Q, F427A blocked cell killing by LFNDTA, translocation across
the plasma membrane, and permeabilization of cells. It did not block conversion of PA63 to an SDS-resistant state, however, or
pore formation in planar lipid bilayers.
One possible explanation of the phenotypes of PA-K397A and PA-D425A is
that their respective cationic and anionic side chains interact during
the transition to the pore. However, we found that the double mutant in
which the side chains at the two sites had been exchanged
(K397D/D425K) was no more active in this transition than the
single mutants (Table I). The same was true of a quadruple mutant
involving exchange of two Lys residues in
27-2
8 for two adjacent Asp residues in
2
10-2
11 (K395D/K397D/D425K/D426K).
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DISCUSSION |
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In the course of mutating residues observed on the luminal aspect of the PA63 heptamer crystal structure, we found three sites at which substitutions caused a major disruption in LFNDTA-mediated cell killing: Lys-397, Asp-425, and Phe-427. The mutations tested at these sites are unlikely to have caused general misfolding, given that the residues are solvent-exposed and did not affect the tryptic and chymotryptic cleavage patterns or expression of the protein. Furthermore, the mutations are highly specific in the functions they affect.
The mutant forms of PA could be activated normally by trypsin, and the resulting PA63 fragments were unimpaired in ability to bind ligands. We demonstrated ligand binding on cells using radiolabeled LFN and in solution by monitoring formation of a characteristic LFN complex. The mutant forms of PA63 were also unimpaired in oligomerization, as shown by the formation of the LFN·PA complex, by retention of chromatographic characteristics, and by electron microscopy. Functional lesions were observed in translocation, as shown by an assay for low-pH-triggered transfer of LFN across the CHO-K1-cell plasma membrane and in pore formation in these cells. These results suggest that mutations in Lys-397, Asp-425, and Phe-427 interfere specifically with the ability of receptor-bound (PA63)7 to undergo conformational changes required for translocation.
Whereas mutations in Lys-397 and Asp-425 blocked pore formation in both cells and planar bilayers, F427A affected this process only in cells. A difference in the pH dependence of pore formation between cells and planar bilayers was observed earlier (6, 27, 28), suggesting a possible effect of the PA receptor on this process.
Mutations at positions 397 and 425 (except for K397Q and K397R) also differed from F427A in their effects on the ability of PA63 to form an SDS-resistant heptamer. Heretofore we used formation of an SDS-resistant heptamer as an apparent indicator of conversion of PA63 prepore to the pore (23). Milne et al. (14) had shown that an SDS-resistant high molecular weight PA band forms after PA activation, endocytosis, and trafficking to the endosome. Lysosomotropic agents or inhibitors of endocytosis blocked formation of the band, indicating that it represents a form generated under acidic conditions. Furthermore, a shift to this band was observed when the cells were treated at a pH known to induce pore formation.
Recently, however, we discovered that the crystallographic
PA63 heptamer is also SDS-resistant. Although purified
oligomeric PA63 is dissociable by SDS if maintained at
pH > 8.0, the crystallization buffer (50 mM Tris-HCl,
pH 8.0-8.5; 20-40 mM CaCl2, 2% polyethylene glycol 8000) induces SDS resistance, even in the absence of
crystals.4 This suggests that
two SDS-resistant forms of (PA63)7 may exist: the pore and the prepore-like form whose structure was determined. In
light of these findings, it is not possible to give an unequivocal interpretation of the changes caused by the mutations at positions 397 and 425. Nonetheless, the inability of mutants to convert to an
SDS-resistant form suggests that this property could be requisite for
pore formation. It is clearly not sufficient, however, because
PA-K397Q, PA-F427A, and PA in which the
22-2
3 has been deleted can all form
SDS-resistant heptamer, despite their inability to form pores in the
plasma membrane.
Precisely how mutations in Lys-397, Asp-425, and Phe-427 block
conformational changes leading to pore formation and translocation is
not evident. According to the model of Petosa et al. (15), for the 22-2
3 loops to form a
transmembrane
-barrel domain 2 must undergo a major conformational
rearrangement involving peeling out of the two flanking
-strands
from the domain. The 2
7-2
8 and
2
10-2
11 loops, where Lys-397, Asp-425,
and Phe-427 reside, are on the opposite side of domain 2 from the
2
2-2
3 loop, and it is unclear how they
would participate in this rearrangement. The elegant analysis of pore
formation by staphylococcal
-hemolysin and the related protein LukF,
showing intersubunit contacts via the N-terminal latch, provides an
example of how such interactions can occur in a pore-forming toxin (21,
29). Dissimilarities in the fold prevent direct application of this
model to PA, however.
Our data show that the requirement for Asp at position 425 is highly specific and the positive charge of Lys-397 is important, implying that these residues form specific contacts with sites on the same or neighboring subunits of the heptamer. Phe-427 presumably forms a hydrophobic contact near that of Asp-425. A double mutation (K397D/D425K) and a quadruple mutation (K395D/K397D/D425K/D426K) were constructed to probe the possibility that Lys-397 might interact with Asp-425, or alternatively Lys-395 and Lys-397 might interact with Asp-425 and D426. The results were negative but do not disprove that such interactions may occur, because the relevant residues in the mutants were out of their normal contexts.
Examination of PA-homologues (component-II of Clostridium botulinum C2 toxin (30), Sb component of C. spiroforme toxin (31), CdtB from C. difficile (32), and Ib component of iota toxin produced by C. perfringens (33)) shows the residues identified here (Lys-397, Asp-425, and Phe-427) to be highly conserved. Both Asp-425 and Phe-427 are absolutely conserved, whereas Lys-397 in PA corresponds to Gln in all of the other proteins. The lack of variation at Asp-425 and Phe-427 further supports the functional importance of these specific residues.
Although we do not yet understand the mechanistic relationship between
pore formation and translocation, the fact that mutations at Lys-397,
Asp-425, and Phe-427 blocked both functions (in native membranes)
strengthens the correlation between these two functions. Mutations at
these sites are likely to be useful in dissecting and interrelating the
processes. Such mutations may also be useful in developing new
pharmaceuticals to treat anthrax. In work to be reported
elsewhere5 we have shown that
the single mutation, F427A; the double mutation, K397D/D425K; or
deletion of the 22-2
3 loop endows PA with
the property of being dominant negative. That is, PA63
derived from either of these mutants co-oligomerizes with wild-type
PA63, but the resulting hetero-oligomers are unable to
mediate translocation. These mutant forms of PA may therefore be useful
as therapeutics for use in neutralizing anthrax toxin in individuals
infected with Bacillus anthracis. Also, because the
mutations alter the structure of PA only in minor ways, its
immunoprotective properties are likely to be preserved, implying that
the mutants could also be used for vaccination.
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ACKNOWLEDGEMENTS |
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We thank Borden Lacy, Jeremy Mogridge, Michael Mourez, and Alan Finkelstein for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R37-AI22021 (to R. J. C.), T-32-GM-07288 (to S. N.), and GM-29210 (to Alan Finkelstein).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Has financial interest in AVANT Immunotherapeutics, Inc. To whom correspondence should be addressed: Tel.: 617-432-1930; Fax: 617-432-0115; E-mail: jcollier@hms.harvard.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008309200
2 J. Mogridge, M. Mourez, and R. J. Collier, J. Bact., in press..
3 J. Mogridge, M. Mourez, and R. J. Collier, unpublished results.
4 D. B. Lacy and R. J. Collier, unpublished results.
5 B. Sellman, M. Mourez, and R. J. Collier, manuscript submitted.
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ABBREVIATIONS |
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The abbreviations used are: ATx, anthrax toxin; PA, protective antigen; nPA, proteolytically nicked PA; PA63, C-terminal 63-kDa fragment of PA; PA20, N-terminal 20-kDa fragment of PA; LF, lethal factor; LFN, N-terminal 255 amino acids of LF; EF, edema factor; CHO, Chinese hamster ovary; DTA, the A moiety of diphtheria toxin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; CHES, 2-(cyclohexylamino)ethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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