(Received for publication, November 27, 1995; and in revised form, January 30, 1996)
From the
The pore-forming -toxin from Staphylococcus aureus is secreted as a soluble monomeric protein. In order to form a
transmembrane channel, the protein has to undergo oligomerization and
membrane insertion. Previous studies have shown that channel formation
is favored by acidic pH. We have analyzed the effect of pH on the
kinetics of channel formation as well as on the conformation of the
toxin. Using a variety of spectroscopic probes for protein structure,
we have shown that
-toxin unfolded upon acidification and that the
unfolding process occurred in at least three steps. The various steps
could be selectively affected by modifying the salt concentration or
the temperature. This unfolding was, however, only partial as the
secondary structure remained native-like as witnessed by far UV CD
measurements. The first unfolding step, corresponding to a region of
the C-terminal half of the toxin, is of particular importance as it
coincided with the exposure of hydrophobic patches on the surface of
the protein as well as with the onset of channel formation. Our
observations strongly suggest that transition of the C-terminal half of
-toxin to a molten globule-like state is required for channel
formation.
-Toxin is a 33-kDa protein secreted by most Staphylococcus aureus strains and is thought to be largely
responsible for the pathogenesis of this bacterium in mammals (for
review, see (1) and (2) ). The toxin has been shown to
irreversibly damage the membranes of a great variety of cells including
erythrocytes from different species(3) , human platelets,
endothelial cells, and mouse adrenocortical Y1 cells.
-Toxin is
secreted as a soluble monomeric polypeptide that can bind to yet
unidentified receptors on the surface of sensitive
cells(2, 4) . Lateral diffusion of membrane-bound
monomers leads to the formation of non-lytic heptameric pore precursors
that are thought to undergo a second conformational change in order to
form the functional transmembrane channel(5, 6) .
Since no crystal structure of -toxin has yet been obtained, the
available structural information is derived from biochemical and
biophysical studies. The protein is mainly composed of
-sheet as
shown by circular dichroism (5, 7) and is in agreement
with secondary structure predictions(8) . Conformational
analysis by limited proteolysis has suggested that the
-toxin
monomer is composed of two domains, corresponding roughly to the two
halves of the protein, separated by a protease-sensitive, glycine-rich
loop(5, 9, 10, 11) . This hypothesis
was reinforced by Bayley and co-workers (12) who have shown, by in vitro fragment complementation experiments, that
cotranslation of the N- and C-terminal halves of the toxin leads to a
product with significant hemolytic activity.
Very little is known
about the mechanism by which -toxin inserts into a membrane. It
has been shown that insertion requires proper
oligomerization(13) , that the glycine-rich loop penetrates
into the bilayer (14) and lines the lumen of the final
channel(15, 16, 17, 18) , and that
channel formation is favored at acidic pH(19, 20) . In
this paper we have analyzed in detail the kinetics of channel formation
by
-toxin in artificial liposomes upon acidification. We show that
the toxin is very sensitive to the local pH at the surface of the
membrane rather than to the bulk pH. Finally, we have made use of these
pH effects to study the structure of
-toxin. Our data provide new
insights into the multidomain organization of
-toxin and suggest
that the protein must partially unfold for channel formation to occur.
Having established that liposome
fusion did not occur, we studied the kinetics of channel formation by
monitoring the kinetics of release of chloride from vesicles containing
the chloride-sensitive dye SPQ. We first checked that chloride efflux
occurred in a dose-dependent manner and that it could be inhibited by
zinc, which has been shown to block the -toxin channel (30) (data not shown). Kinetics were then measured at various
pH values with liposomes having different lipid compositions. Fig. 1, A and B, shows that the kinetics
obtained using an EPC/EPA (95:5%) mixture and pure EPG were very
sensitive to pH.
Figure 1:
Effect of pH on the -toxin-induced
chloride efflux from SPQ-containing liposomes. Chloride efflux from
liposomes containing 95% EPC and 5% EPA (A) or 100% EPG (B) was measured at various pH values (25 °C). The time
point at which
-toxin was added (80 ng/ml) is indicated by the arrow. a.u., arbitrary units. C, the maximal
rate of
-toxin-induced chloride efflux was plotted as a function
of bulk pH. Efflux from pure EPG vesicles was also plotted as a
function of the local pH at the membrane
interface.
When plotting the maximal rate of chloride efflux as a function of pH, it clearly appears that chloride efflux occurred at far lower pH values for EPC than for EPG vesicles; the EPC/EPG (50:50) vesicles adopted an intermediate behavior (Fig. 1C). Chloride efflux could only be observed from EPC vesicles at pH values below 4, whereas for EPG vesicles, efflux readily occurred at pH 5.5. A decrease in the rate of chloride efflux was observed when the pH was further lowered (data not shown). This inhibition occurred at pH values below 4.5, 4.2, and 3 for pure EPG, EPC/EPG (50:50), and pure EPC vesicles, respectively.
We then determined whether the differences in kinetics observed for the various types of liposomes at the different pH values may reflect the amount of oligomers bound to the vesicles. Therefore, at the end of each chloride efflux measurement, the vesicles were recovered by ultracentrifugation and analyzed by SDS-polyacrylamide gel electrophoresis under non-boiling conditions. Remarkably similar amounts of oligomers could be seen at all pH values, whether efflux had occurred or not, confirming the existence of non-lytic oligomers (10, 31) (Fig. 2). The amounts of oligomer were also very similar for all the lipid compositions studied (data not shown). There seemed to be no correlation between the amount of oligomers and the chloride efflux nor did pH seem to have a significant effect on the oligomerization process.
Figure 2:
Effect of pH on the amount of -toxin
oligomers bound to EPC/EPA vesicles. After chloride efflux
measurements, vesicles were sedimented by ultracentrifugtion,
solubilized in sample buffer, and run on an SDS-polyacrylamide gel. To
prevent disruption of the oligomer, samples were not boiled. HMW, high molecular weight standards (200,000, 116,250,
97,400, 66,200, 45,000); LMW, low molecular weight standards
(97,400, 66,200, 45,000, 31,000).
The apparent preference at a
given pH of -toxin for EPG could thus be explained only by a high
affinity of the toxin for the phosphatidylglycerol head group or for
negatively charged head groups in general. Alternatively,
-toxin
may be affected by the pH at the surface of EPG vesicles. A reduced
local pH is expected since the negatively charged head groups give rise
to an electrical surface potential that in turn decreases the pH at the
membrane surface(32, 33) . The
pH between the
bulk and the vesicle surface depends on the surface charge density of
lipids, on the local anion concentration, as well as on the size of the
vesicles. The Gouy-Chapman theory predicts a
pH of 2.7 between a
low ionic strength solution and a planar lipid bilayer composed of 100%
negatively charged lipids. We have measured the pH near the surface of
the EPG vesicles under our experimental conditions. For example, for a
bulk pH of 5, the pH at the membrane surface was 1.47 pH units lower
than the bulk pH, which is in close agreement with our previous
measurements(25) . When the maximal velocities of chloride
efflux are expressed as a function of interfacial pH (Fig. 1C), it appears that the onset of efflux occurs
at a similar pH for EPG and EPC vesicles, demonstrating that the toxin
is sensitive to local pH rather than to the chemical structure of the
lipid head group.
Figure 3:
Distribution of aromatic residues along
the -toxin sequence.
, tryptophan residue;
, tyrosine
residue; *, phenylalanine residue.
Figure 4:
Effect of pH on the near UV CD spectrum of
-toxin. The measurement of the near UV CD spectrum of
-toxin
(0.44 mg/ml) was taken at various pH values (A, 50 M NaCl). B, the intensity of the peak at 281 nm was plotted
as a function of pH for two different salt concentrations (25
°C).
This
change in near UV CD signal was partially reversible. Indeed, 73% of
the signal at 281 nm could be recovered when -toxin was first
incubated at pH 3.2 and then diluted into a buffer (50 mM NaCl, 50 mM citric acid/Na
HPO
) at
pH 7. However, at the high concentration required for near UV CD, an
incubation of
-toxin at pH 3.2 for several minutes led to
aggregation upon neutralization.
To establish whether this
acid-induced flexibility of the protein was accompanied by a loss of
secondary structure, the far UV CD spectrum of -toxin was measured
at various pH values. As shown in Fig. 5, the spectrum is rather
insensitive to pH. In particular, the ellipticity at 210 nm did not
gradually decrease upon acidification indicating that there was no
significant loss in secondary structure. Only for pH values below 2.5
and at 50 mM NaCl could we detect a significant contribution
by random coil elements. In Fig. 5, a slight increase in the
ellipticity can be observed upon acidification, but as the far UV CD
spectra of
-sheet proteins are very sensitive to the
environment(35) , the appearance of the small changes observed
is beyond interpretation. The observed variations are probably due to
changes in the conformation of aromatic residues that are known to
contribute significantly to the far UV CD spectrum and especially to
that of
-sheet proteins(34, 36) .
Figure 5:
Effect of pH on the far UV CD spectrum of
-toxin. The measurement of the far UV CD spectrum of
-toxin
(0.66 mg/ml) was taken at different pH values (50 M NaCl, 25
°C).
Figure 6:
Effect of pH on the maximal emission
wavelength of tryptophans. The maximal emission wavelength of
tryptophans was determined at various pH values and at four different
NaCl concentrations: , 3 mM;
, 50 mM;
, 100 mM; and
, 150 mM (25 °C). Inset, the total fluorescence (
) corresponding to the
area of the tryptophan emission spectrum was plotted as a function of
pH (150 mM NaCl). The pH dependence of
in
the presence of 150 mM NaCl (
) was plotted again in
order to facilitate the comparison. a.u., arbitrary
units.
We have also investigated the effect of
temperature on the acid denaturation of -toxin. When raising the
temperature to 37 °C, the first unfolding step observed in the
presence of 150 mM NaCl occurred at 0.5 pH unit higher than at
25 °C. Indeed, at 25 °C,
shifted to 333.5
± 0.4 nm at pH 3.7; however, at 37 °C, a similar
(
= 333.2 ± 0.1
nm) was already observed at pH 4.3. The second unfolding step was not
significantly affected by this temperature shift.
Figure 7:
Binding of the hydrophobic dye ANS to
-toxin at various pH values. 50 µM ANS was added to a
solution containing 35 µg/ml
-toxin, 150 mM NaCl, and
20 mM buffer at the desired pH; and its fluorescence at 480 nm
(
) was measured. The curve corresponding to the maximal emission
wavelength of tryptophans (
) is that of Fig. 6. a.u., arbitrary units.
The increase in hydrophobicity observed upon acidification
was observed at all salt concentrations studied. The hydrophobic
intermediate was, however, stabilized over a wider pH range in the
presence of 150 mM NaCl. The effect of temperature on ANS
binding was also analyzed. The appearance of the hydrophobic
intermediate occurred at higher pH at 37 °C than at 25 °C (data
not shown) in a manner similar to that observed when measuring
. These observations indicate that the increase in
hydrophobicity is concomitant with the first unfolding step.
Similar
experiments were performed using an excitation wavelength
() of 295 nm instead of 380 nm. Under these
conditions, tryptophan residues are excited and ANS can only be excited
through energy transfer. The results were very similar to those
obtained for
= 380 nm suggesting that ANS
binds in close proximity to the tryptophan residues, most of which are
clustered in the C-terminal half of the toxin.
The present study on the effect of pH on the conformation of
-toxin, as well as its ability to form channels in artificial
membranes, has led to new information on the three-dimensional
structure of the toxin and on the mechanism of membrane insertion.
Our data would be consistent with the
following multidomain organization of -toxin. The N-terminal half
of the protein would form a single domain (domain 1) separated from the
C-terminal region by the so-called glycine-rich loop. This domain 1
would be the less pH-sensitive region of
-toxin, and the presence
of salts would completely protect it toward acidic unfolding. In
contrast, the C-terminal region would be very sensitive to pH. Upon
acidification, it would unfold in a two-step process (Fig. 6).
The fact that the two-step process could be selectively affected by
varying the salt concentration or the temperature suggests that the
C-terminal region might be formed by two domains. This, however,
remains to be confirmed.
One must keep in mind that all mentioned unfolding steps are only partial as the secondary structure is essentially maintained under the different conditions. It seems that upon acidification, the various domains sequentially undergo a transition to a molten globule-like state. The most interesting folding intermediate was obtained, at pH 3.5, at physiological salt concentrations. In this folding intermediate, domain 1 is probably still fully folded, but the rest of the protein shows typical characteristics of the molten globule-like state: native-like secondary structure, increased flexibility of the tertiary structure, and binding of ANS. However, this folding intermediate does not seem as compact as most molten globule-like states since some tryptophans appear to be accessible to the solvent.