(Received for publication, June 2, 1995; and in revised form, September 8, 1995)
From the
The adenylate cyclase (CyaA) secreted by Bordetella
pertussis is a toxin that is able to enter eukaryotic cells and
cause a dramatic increase in cAMP level. In addition, the toxin also
exhibits an intrinsic hemolytic activity that is independent from the
ATP cycling catalytic activity of the toxin. Both the cytotoxic and
hemolytic activities are calcium-dependent. In this work, we have
analyzed the calcium interacting properties of CyaA. We have shown that
CyaA exposed to CaCl could retain membrane binding
capability and hemolytic activity when it was further assayed in the
presence of an excess of EGTA. Determination of the calcium content of
CyaA exposed first to calcium and subsequently to EGTA indicated that
some (3, 4, 5) calcium ions remained bound to
the protein, suggesting the existence of Ca
binding
sites of high affinity. Binding of Ca
to these sites
might be necessary for both the membrane binding capability and the
hemolytic activity of the toxin. In addition, CyaA possesses a large
number (about 45) of low affinity (K
= 0.5-0.8 mM) Ca
binding sites that are located in the C terminus of the toxin,
between amino acids 1007 and 1706. This region mainly consists of about
45 repeated sequences of the type
GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX
(Repeat in ToXin) bacterial protein family. Our data suggest that each
one can bind one calcium ion. Circular dichroism spectroscopy analysis
showed that calcium binding to the low affinity sites induces a large
conformational change of CyaA, as revealed by an important increase in
the content of
-helical structures. This conformational change
might be directly involved in the Ca
-dependent
translocation of the catalytic domain of CyaA through the plasma
membrane of target cells.
One of the essential virulence factors of the whooping cough
agent, Bordetella pertussis, is the secreted adenylate cyclase
toxin
CyaA()(1, 2, 3, 4) . It
is a protein of 1706 residues that is converted to the active toxin by
a post-translational palmitoylation of an internal lysine
residue(5) . This modification requires the product of the
accessory cyaC gene(6) . The toxin is a bifunctional
protein. Its purified 177-kDa toxic form exhibits both invasive
adenylate cyclase (cytotoxic) and hemolytic (pore-forming)
activities(7, 8, 9, 10, 11, 12) .
The adenylate cyclase (AC) catalytic domain of CyaA resides in the 400
amino-proximal residues and its enzymatic activity requires eukaryotic
calmodulin (CaM)(13, 14, 15, 16) .
The toxin delivers the AC domain directly across the plasma membrane of
a variety of epithelial and immune effector cells and disrupts their
physiological functions by uncontrolled synthesis of
cAMP(11, 17, 18, 19, 20) .
The C-terminal 1330 residues of CyaA are required for cell targeting
and delivery of the AC-domain into cells (8, 10, 11) and can act independently as a
hemolysin(12, 21, 22) . Interestingly, no
dedicated target cell binding domain could be identified within this
hemolysin portion of CyaA. The structural integrity of CyaA appears to
be essential for both delivery of the AC-domain into target cells and
the pore-forming activity of the toxin. (
)The C-terminal
part of CyaA (residues 400-1706) exhibits several features common to
all RTX (Repeat in ToXin) proteins(24) . It contains four
hydrophobic stretches (from residues 500-700) involved in
formation of the cation-selective CyaA
channels(25, 26) , the target site for the
post-translational modification of CyaA (Lys residue 983, which is
palmitoylated)(5) , and 40-45 glycine and aspartate-rich
nonapeptide repeats (from residues 1015 to 1613) of the prototype
GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX
bacterial protein family(14) .
CyaA is a calcium-binding
protein that undergoes conformational changes upon binding of
calcium(18, 19) . The repeat motifs of CyaA (between
residues 1007 and 1612) are suspected to be involved in calcium
binding, as it was shown that deletion of similar repeats abolished
calcium binding to the -hemolysin (HlyA) of Escherichia
coli(27, 28, 29) . Indeed the repeat
motifs, GGXGXDXLX, were found, in
the three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa, to constitute a new type of
calcium-binding structure(30) . The cytotoxic activity of CyaA
is absolutely calcium-dependent; the translocation of the AC catalytic
domain across target cell membranes occurs only at calcium
concentrations above 0.1 mM. However, the calcium requirement
of the hemolytic activity is still a matter of controversy (8, 12) .
In this study, we have analyzed the calcium binding properties of CyaA and shown that CyaA harbors probably two classes of calcium binding sites of different affinities. Binding of calcium to a small number of high affinity binding sites might be necessary for the hemolytic activity of the toxin. In addition, CyaA contains about 40-45 low affinity calcium binding sites, which are located in the repeat region of the toxin. Binding of calcium to these low affinity sites induces major structural rearrangements of CyaA that may be involved in delivery of the AC domain into target cells.
Figure 1:
Tightly
bound calcium is required for cell binding and hemolytic activity of
CyaA. Two disposable desalting columns packed with 10 ml of Sephadex
G-25 (PD-10; Pharmacia) were equilibrated with 20 mM Tris-HCl,
pH 8.0, 150 mM NaCl, 0.1% bovine serum albumin, containing
either 1 mM CaCl (TNBC) or 2 mM EGTA
(TNBE). Samples of 0.25 ml of CyaA stock solution (0.3 mg/ml; in 20
mM Tris-HCl, pH 8.0, 8 M urea, and 2 mM
EGTA) were applied to each of the two columns. Upon elution at room
temperature with either TNBC or TNBE buffer, the two renatured
calcium-loaded and calcium-free toxin samples were collected (1.7 ml)
and adjusted to equal toxin concentrations (17 units/ml) with the
corresponding elution buffer. Toxin activities were tested at final
concentration of 2.4 units/ml, using sheep erythrocytes (5
10
cells/ml) resuspended in 20 mM Tris-Cl, pH 8.0,
150 mM NaCl, and either 2 mM CaCl
, or 2
mM EGTA. Internalized (hatched bars) and cell-bound (gray bars) AC activity were determined after 30 min of
incubation at 37 °C (A), and the extent of erythrocyte
lysis (B) was determined at the indicated times, as described
under ``Materials and Methods.'' The error bars indicate the standard error mean of two independent
experiments.
To test
this hypothesis, we attempted to measure the calcium content of the
toxin renatured either in the presence or in the absence of
Ca and exposed subsequently to the calcium-chelator
EDTA. The toxin, purified in the presence of 8 M urea and 2
mM EDTA, was renatured, as described above, by chromatography
on Sephadex G-25 columns equilibrated in buffer A containing either 2
mM CaCl
or 5 mM EDTA. The renatured
protein was then submitted to two subsequent chromatographies on G-25
columns equilibrated in buffer A supplemented with 5 mM EDTA.
The calcium content of the proteins, recovered after the third G-25
column, was then analyzed by atomic absorption spectrophotometry.
Between three and five calcium atoms were bound per CyaA molecule when
the toxin had been renatured in the presence of 2 mM
CaCl
. Less than 1 calcium ion was bound per CyaA molecule
(from 0.6 to 0.8), when the toxin had been renatured in the presence of
5 mM EDTA. Unfortunately, we could not assay the membrane
binding capacity and the hemolytic activity of the samples subjected to
the atomic absorption analysis. In fact, at the high concentrations of
toxin that were used in these experiments (4 mg/ml, 22 µM)
in order to get a reliable measurement of the calcium content, CyaA had
a tendency to aggregate, which diminished its biological activities
substantially.
Altogether, however, these results suggest that when CyaA is exposed to calcium during its renaturation, it binds some calcium ions that could not be removed during a subsequent exposure of the toxin to EDTA or EGTA. These bound calcium ions are necessary and sufficient for the membrane binding capability and the hemolytic activity of CyaA, but insufficient for its cytotoxic activity.
Figure 2:
Schematic representation of the CyaA
derivatives that were examined for binding of calcium. The numbers that
follow the symbol in the names of the corresponding expression
plasmids mark the positions of the first and the last codons of the
deleted parts of cyaA reading frame. The various subdomains of
the wild-type CyaA are marked by hatched areas and are
delimited by the numbers of the flanking residues. The shaded areas represent the regions that are missing from the truncated CyaA
proteins.
Figure 3:
Localization of calcium binding sites of
CyaA by Ca
overlay. A, SDS-PAGE
analysis of the purified wild-type CyaA and of the truncated
derivatives. B,
Ca
overlay. The
purified proteins were separated by SDS-PAGE (7.5% gel),
electrotransfered to nitrocellulose membrane, and probed for calcium
binding with 2 µM CaCl
solution containing
Ca
(2 µCi/ml). The membrane was then
washed, dried, and exposed for autoradiography. The autoradiogram shown
was obtained after 3 days of exposure. C, Ponceau S staining
of the membrane-bound proteins. The analyzed proteins are encoded by
the following plasmids: lane 1, pCACT3; lane 2, pACT; lane 3, pACT
C217; lane 4 pACT1007; lane 5, pACT863; lane 6, pACT
1008-1490; lane 7, pACT
622-1489; lane 8, pACT
827-887; lane 9, pACT
622-779; lane 10, pDIA5240.
Figure 4:
Calcium binding properties of CyaA and
RD-CyaA. Calcium binding to CyaA (13 µM, ), RD-CyaA
(17 µM,
), and ACT1007 (15 µM,
)
was determined by ultrafiltration, as described under ``Materials
and Methods.'' Similar data were obtained in three different
experiments.
Figure 5:
Far UV CD spectra of CyaA and RD-CyaA in
the absence and in the presence of calcium. CD spectra of CyaA (A) and RD-CyaA (B) in the absence (<0.1 mM CaCl, plain line) or in the presence of
calcium (2 mM CaCl
, dashed line), were
recorded as described under ``Materials and Methods.'' Each
spectra represents the average of 5 scans, carried out at 20 °C
between 180 and 260 nm, using 0.1-nm steps and an integration time of 2
s. C, calcium dependence of the mean differential residual
molar circular dichroic extinction coefficient (
in M
cm
) at 220 nm of
CyaA (
) and of RD-CyaA (
). The error bars indicate
the standard error mean of four independent
titrations.
The results presented here suggest that CyaA harbors two
classes of calcium binding sites. The first class might consist of a
small number (3, 4, 5) of high affinity
Ca binding sites, whereas the second class consists
of about 45 calcium binding sites of low affinity. Calcium binding to
the high affinity sites appears to be critical for the membrane binding
capability and the hemolytic activity of the toxin, while calcium
binding to the low affinity sites could be involved in the cytotoxic
activity of CyaA, that is, in the translocation of the catalytic domain
through the membrane of the target cells.
The presence of high
affinity calcium binding sites in CyaA is suggested by two sets of
results. First, we have shown that CyaA, which had been renatured in
the presence of calcium, exhibited membrane binding capability and
hemolytic activity when it was further assayed in the presence of an
excess of EGTA, whereas CyaA, which had been renatured in the absence
of calcium, did not. These findings explain the apparent discrepancy
between our previous data (8) and those of Rogel et
al.(12) . Indeed, Rogel et al.(12) observed a calcium-independent hemolytic activity of CyaA
when it had been renatured in the presence of CaCl. In
contrast, we showed an absolute calcium requirement for the hemolytic
activity of the toxin that was renatured in the absence of
calcium(8) . Second, when CyaA had been renatured in the
presence of calcium and subsequently exposed to the calcium chelator
EDTA, 3-5 calcium ions remained bound to the protein, as
determined by atomic absorption spectrophotometry. Altogether, these
data suggest that (i) CyaA binds a few calcium ions that cannot be
removed by addition of EGTA or EDTA without denaturation and (ii) these
bound calcium ions are required for the membrane binding and hemolytic
activities of the toxin.
A more precise determination of the number,
and affinities of these putative high affinity calcium binding sites
proved to be very difficult, essentially because CyaA had a tendency to
aggregate at protein concentrations required to achieve reliable
determinations of the bound calcium. Hence, direct measurements, on the
same samples, of both the amount of calcium bound to the protein
(assessed by atomic absorption) and the membrane binding capability and
hemolytic activity of CyaA (measured in the presence of EGTA) could not
be performed. However, our present data clearly indicate that there are
only a small number of these potential high affinity calcium binding
sites and that they should have a much higher affinity for calcium than
EDTA or EGTA (that is, an equilibrium constant in the nanomolar range).
At present, we do not know the localization of these potential high
affinity calcium binding sites. It appears that they are not located in
the last 700 residues of CyaA as the truncated derivative, RD-CyaA, did
not exhibit tightly bound calcium. ()However, one cannot
exclude that the folding of the Asp-Gly-rich repeat region is somewhat
different in RD-CyaA as compared with the full-length toxin. It is
interesting to recall, that the
-hemolysin of E. coli also exhibited a Ca
-independent hemolytic
activity if it had been previously exposed to calcium
ions(29) . It will be interesting to determine whether HlyA
also possesses high affinity calcium binding sites.
The second class
of calcium binding sites of CyaA is represented by a large number
(about 45) of low affinity sites, that bind Ca in the
submillimolar range (K
= 0.5-0.8
mM). These sites are located in the Asp-Gly rich repeat region
of CyaA, at the C terminus. These repeats, the prototype of which is
GGXG(N/D)DX(L/I/F)X, are common to several
different proteins of the RTX family(24) . Previous studies,
carried out on E. coli
-hemolysin, have suggested that
these repeated sequences could be involved in calcium
binding(27, 28, 29) . The recently reported
three-dimensional structure of the alkaline protease of P.
aeruginosa, which possesses a small number of such Asp-Gly-rich
motifs, indicates that, indeed, these repeats constitute a novel type
of calcium binding sites(30) . The nonapeptide repeats are
organized in a
-roll, which consists of two opposing sheets of
paralleled
-strands. The
-strands comprise the last 5 amino
acids of each nonapeptide sequence ((N/D)DX(L/I/F)X)
and are connected by loops derived from the first 4 amino acids of the
repeated sequence (GGXG). Ca
is
hexacoordinated between two adjacent loops of the
-roll, and as a
whole, each repeat binds one calcium ion. Among the RTX protein family,
CyaA contains by far the largest number of these Asp-Gly rich motifs,
40-45, depending on the consensus criteria used for their
computation(14) . Our present results suggest that in solution,
each repeat motif of CyaA is able to bind one calcium ion. However, in
contrast to the tight Ca
binding to the repeat motifs
of alkaline protease(30) , the majority of the repeat motifs of
CyaA exhibited only low affinity for calcium. This is not unexpected as
most of the repeat motifs of CyaA do not perfectly match the
GGXGXDXLX consensus.
The
secondary structure composition of RD-CyaA, as deduced from the CD
spectroscopy studies, strongly suggests that the repeated sequences of
CyaA are structurally organized, like those of P. aeruginosa alkaline protease, in a small -strand followed by a
connecting loop. The CD analysis indicates that the secondary structure
of RD-CyaA consists, in the presence of calcium, of 35% of
-structures. This is roughly the value one would expect if the
first 5 amino acids of each nonapeptide repeats were in a
-strand
conformation. The fact that the addition of Ca
increases the
-sheet content of RD-CyaA indicates that
binding of calcium to the repeat motifs stabilizes this secondary
structure.
Interestingly, inspection of the amino acid sequence
revealed a striking peculiarity of the repeat region of CyaA as
compared with other RTX proteins(24) . The nonapeptide repeats
of CyaA are arranged in five groups of 8-10 repeats, separated by
20-30-residue-long unrelated sequences. This modular organization
is unique to CyaA, as all of the other RTX proteins have only one group
of 8-13 repeats. The ``intervening'' sequences consist
essentially of short stretches of -helix-forming amino acids, that
are interrupted by Gly and Pro residues. Our CD studies indicate a
large increase in the
-helical content of RD-CyaA upon
Ca
binding to the low affinity sites. We suggest that
the intervening sequences, which bridge the subgroups of repeats,
undergo a structural rearrangement as a consequence of Ca
binding to CyaA, and shift from an extended conformation to an
-helical one. Hewlett et al.(19) have previously
observed, by electron microscopy, a conformational change of CyaA upon
binding of calcium. The present study extends the previous observation
and identifies the C-terminal part of CyaA as the main
calcium-responsive domain of the molecule at millimolar calcium
concentrations. On the other hand, it is well established that
millimolar concentrations of free Ca
are required for
the cytotoxic activity of CyaA. It is thus tempting to speculate that
the conformational change that occurs in the repeat region of CyaA upon
calcium binding is directly involved in the
Ca
-dependent translocation of the catalytic domain of
CyaA through the plasma membrane of target cells.