(Received for publication, August 26, 1996, and in revised form, December 6, 1996)
From the Département de Biochimie,
Université de Genève, 30 quai E. Ansermet,
1211 Genève, Switzerland and the § Microbiology and
Tumorbiology Center, Karolinska Institutet,
17177 Stockholm, Sweden
Conformational changes occurring upon membrane
binding and subsequent insertion of staphylococcal -toxin were
studied using complementary spectroscopic techniques. Experimental
conditions were established where binding could be uncoupled from
membrane insertion but insertion and channel formation seemed to be
concomitant. Binding led to changes in tertiary structure as witnessed
by an increase in tryptophan fluorescence, a red shift of the
tryptophan maximum emission wavelength, and a change in the near UV CD
spectrum. In contrast to what was observed for the soluble form of the
toxin, 78% of the tryptophan residues in the membrane-bound form were accessible to the hydrophilic quencher KI. At this stage, the tryptophan residues were not in the immediate vicinity of the lipid
bilayer. Upon membrane insertion, a second conformational change
occurred resulting in a dramatic drop of the near UV CD signal but an
increase of the far UV signal. Tryptophan residues were no longer
accessible to KI but could be quenched by brominated lipids. In the
light of the available data on channel formation by
-toxin, our
results suggest that the tryptophan residues might be dipping into the
membrane in order to anchor the extramembranous part of the channel to
the lipid bilayer.
Despite the importance of membrane damaging proteins such as the
immune proteins C9 of complement and perforin or pore-forming toxins
from pathogenic bacteria, little is known about the precise mechanism
that enables these proteins to convert from a water-soluble protein to
a transmembrane channel. Channel-forming toxins such as staphylococcal
-toxin are extremely useful model proteins to address this issue
since they are relatively easy to obtain in large amounts and will form
channels in vitro.
-Toxin is secreted by Staphylococcus aureus as a soluble
monomeric protein. However, by the time it has reached its final goal,
the toxin has turned into a heptameric transmembrane complex (for
review see Refs. 1, 2). The various steps that allow this metamorphosis
are the following. The toxin binds to an unidentified receptor on the
surface of the target cell where it oligomerizes, thereby leading to a
non-lytic pre-pore complex (3, 4). A second conformational change is
required for insertion of the heptamer into the membrane in order to
form the channel.
Although no crystal structure is yet available for either the soluble
or the heptameric form of the toxin, structural data based on
mutagenesis, biochemical, and biophysical studies are rapidly
accumulating. The soluble monomer is composed of at least two domains
as illustrated by the fact that the protein unfolds in several steps
upon acidification of the medium (5). It has been speculated that the
N- and C-terminal halves of the polypeptide chain may constitute these
domains based mainly on the observation that a central glycine-rich
segment is very sensitive to proteases and most likely forms an
interdomain loop (3, 6-8). Amino acids implicated in membrane binding
were found in both halves of the molecule (9-11). Upon binding, the
central loop becomes protected toward proteases (3, 4, 6), and finally
channel formation involves penetration of this loop into the membrane (4, 8, 9, 12-15). Using single cysteine mutants labeled with an
environment-sensitive probe, Valeva and colleagues (16) have recently
narrowed down the membrane inserting sequence to amino acids 118-124
and suggested that these residues might line the channel. It has been
speculated that the loop might fold into a -hairpin upon membrane
insertion (2, 4). By the contribution of each monomer in the heptamer,
the transmembrane domain of
-toxin would be a 14-stranded
-barrel
resembling the barrel of a porin monomer (17, 18). The existence of
this barrel has recently been confirmed by x-ray crystallography (19).
It is unclear at the present time whether parts of the protein other
than the central loop penetrate into the membrane. Certain amino acids near the C terminus were shown to be accessible to the hydrophobic probe 2-[3H]diazofluorene suggesting that they might
penetrate into the membrane (20), but this remains to be confirmed
using complementary methods.
In the present paper, we have analyzed the conformational changes that
are associated with membrane binding and membrane insertion. Using
brominated phospholipids, we have investigated whether the tryptophan
residues in -toxin reached the vicinity of the hydrophobic core of
the bilayer upon membrane interaction. Brominated lipids have indeed
been useful in determining the topology of membrane proteins (21, 22)
as well as studying the membrane interaction of the pore-forming toxin
colicin A (23-25). Tryptophan fluorescence spectroscopy, circular
dichroism, and protease sensitivity were used to further compare the
structure of the soluble, the membrane-bound, and the membrane-inserted
forms of
-toxin.
-Toxin was produced by strain Wood
46 and purified as described previously (26). The protein concentration
was determined by measuring the absorption at 280 nm based on an OD of
1.8 for a 1 mg/ml solution.
Purified -toxin
was concentrated to about 2 mg/ml and dialyzed overnight against 10 mM NaCl, 20 mM HEPES, pH 7.0, at 4 °C. The
toxin was then incubated at 37 °C for 2 h and vortexed several times. The heptamers were separated from the remaining monomers by gel
filtration on a Sephadex G-75 (Pharmacia Biotech Inc.) column (1.5 cm
in diameter and 70 cm in height) equilibrated with the same buffer.
Large unilamellar liposomes were prepared by reverse phase evaporation as described by Szoka and Papahadjopoulos (27, 28). To calibrate the liposomes in size, the vesicle suspension was extruded through 0.2-µm polycarbonate filters (Nucleopore, Plesanton, CA). Liposomes were either formed of pure dioleoylphosphatidylglycerol (DOPG)1 or of a one to one mixture of DOPG and dioleoylphosphatidylcholine (DOPC). The corresponding lipids brominated at positions 9-10 of the two acyl chains were also used, Br-DOPG and Br-DOPC (Avanti Polar Lipids, Alabaster, AL). Bromide addition was shown not to alter the physicochemical properties of DOPG and DOPC (25). Liposomes were prepared in a buffer containing 150 mM NaCl, 20 mM HEPES, pH 7.4. In order to measure chloride efflux, 1.5 mg/ml 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ, Molecular Probes) was encapsulated into the liposomes as described previously (5) in a buffer containing 100 mM KCl, 20 mM HEPES, pH 7.4.
Fluorescence MeasurementsAll fluorescence experiments were carried out using a PTI spectrofluorimeter with a temperature-controlled sample holder (25 °C unless specified). Chloride efflux and tryptophan fluorescence experiments were carried out as described previously (5).
To monitor the insertion of -toxin into brominated phospholipids,
the excitation and emission wavelengths were set at 295 and 330 nm,
respectively, with 5-nm slit widths.
-Toxin was initially diluted to
6 µg/ml (180 nM) in 150 mM NaCl, 20 mM MES at the desired pH. Liposomes were then added to
reach a final lipid concentration of 0.24 mM, which
corresponds to approximately 1011 liposomes per ml
(considering that the liposomes had an average diameter of 0.2 µm and
that the surface of a lipid head group was 70 Å2).
In order to determine the temperature dependence of membrane insertion and channel formation, the kinetics of quenching of tryptophan fluorescence by Br-DOPG and the kinetics of chloride release were measured at 3 °C intervals between 10 and 40 °C. For each curve, the maximal slope was determined and plotted in semi-log as a function of 1/T.
The amount of -toxin bound to liposomes was quantified as follows.
The tryptophan emission spectrum was measured for
-toxin in
solution; vesicles were then added, and the samples were allowed to
equilibrate for 60 min in order to reach a steady state. The samples
were then centrifuged using a TL100.3 Beckman rotor for 40 min at
60,000 rpm, and the tryptophan fluorescence spectrum of the supernatant
(corresponding to the unbound toxin) was measured. The fraction of
bound toxin was calculated as being (Fsol
Fsup)/Fsol, where
Fsol is the area of the tryptophan emission
spectrum of
-toxin in solution and Fsup the
area of that of the supernatant.
In order to determine the extent of quenching of tryptophan
fluorescence by brominated phospholipids, the area of the tryptophan emission spectrum of -toxin after 60 min incubation with brominated liposomes was compared with that of
-toxin in the absence of liposomes, unless specified otherwise.
In order to determine the extend of tryptophan quenching by KI of
-toxin in the membrane-associated form,
-toxin was first incubated with the desired vesicles at the appropriate pH for 60 min to
allow binding and/or insertion. The vesicles were then pelleted as
described above, resuspended in the appropriate buffer containing
either 150 mM KI or 150 mM NaCl, and the
tryptophan emission spectra were measured.
Circular dichroic experiments were
carried out at room temperature on a Jasco 715 spectrometer. Quartz
cells of 0.1 and 1 cm path lengths were used for measurements in the
far (205-250 nm) and near (260-325 nm) ultraviolet (UV),
respectively. Small unilamellar vesicles were prepared by sonication as
described previously (23) in 150 mM NaCl, 20 mM
MES, at pH 4.75 and pH 5 for DOPG and DOPG/DOPC (50:50, w/w),
respectively. -Toxin (10 µM) was added to the vesicles
(7.5 mM lipids) and allowed to incubate for 60 min at room
temperature. Samples were then briefly sonicated on ice (3 times for
5 s), and their near UV CD spectrum was measured. For far UV CD
measurements, the samples were diluted 2-fold with their respective
buffers. The amount of unbound toxin was determined by comparing the
tryptophan fluorescence spectra of the sample before and after
sedimentation of the vesicles as described above.
Vesicles were prepared as described for
the CD experiments. -Toxin was incubated with the liposomes for 60 min at room temperature prior to addition of the Pronase (1:50,
protease/toxin ratio, w/w). After 30 min of incubation at 37 °C,
sample buffer was added, and samples were boiled unless specified
otherwise. Samples were then analyzed by SDS-PAGE as described by
Laemmli (29). The same cleavage patterns were obtained whether
liposomes had been made by reverse phase evaporation or by
sonication.
We have previously shown that -toxin is
able to form channels in egg phosphatidylglycerol vesicles at acidic pH
(5). As illustrated Fig. 1A,
-toxin is
able to induce chloride efflux from Br-DOPG vesicles in a similar
pH-dependent manner. In parallel experiments, kinetics of
changes in tryptophan fluorescence were measured to investigate whether
brominated lipids were able to quench the intrinsic fluorescence of
-toxin upon membrane insertion. The lipids used had bromines
attached at positions 9 and 10 of the acyl chains. At pH 4.75, where
channel formation occurred (Fig. 1A), the intrinsic
fluorescence dramatically decreased upon addition of Br-DOPG vesicles
(Fig. 1B). Insertion was irreversible since further addition
of non-brominated DOPG vesicles did not lead to dequenching of the
tryptophan fluorescence (not shown). Note that the time scales are
different in Fig. 1, A and B. The differences in
kinetics are due to the fact that one to two channels are sufficient to
empty one vesicle from its chloride content (short time scale, 5);
however, in order to detect quenching of tryptophan fluorescence, a
significant proportion of the total toxin population must insert into
the brominated bilayer (longer time scale). At pH 6 or 7, channels did
not form, and tryptophan fluorescence was not affected by addition of
brominated DOPG vesicles.
These experiments indicate that quenching of the intrinsic fluorescence
by brominated lipids can be used to monitor membrane insertion of
-toxin. Moreover, they show that upon channel formation, tryptophan
residues become accessible to quenchers that are located within the
lipid bilayer. The interpretation of this observation will be discussed
later (see "Discussion").
Having established that quenching of tryptophan residues
by brominated phospholipids can be used to follow membrane insertion of
-toxin, we have investigated whether we could uncouple insertion from channel formation. We were unable to establish experimental conditions where insertion occurred without channel formation. We have
then analyzed the temperature dependence of both membrane insertion and
channel formation into Br-DOPG vesicles (pH 4.75). As shown in Fig.
1C, the temperature dependence was the same for both events.
These observations suggest that the two techniques used here,
fluorescence quenching by brominated phospholipids and release of
entrapped markers, measure the same event and that membrane insertion
and channel formation occurred concomitantly under our experimental
conditions.
The decrease in tryptophan
fluorescence observed upon addition of Br-DOPG vesicles to -toxin at
pH 4.75 indicated that, upon channel formation, certain tryptophan
residues became accessible to quenching by bromine (Fig.
1B). Based on the tryptophan fluorescence spectra shown in
Fig. 2A, the fluorescence was quenched by
60 ± 4% (n = 5). The residual 40% could be due
to a fraction of unbound toxin, a fraction of inaccessible tryptophans,
or to the inefficiency of bromine as a quencher. The first possibility
could be ruled out since all the toxin was bound to the vesicles as
indicated by the fact that the supernatant obtained after sedimentation of the membranes showed no fluorescence (Fig. 2A). In order
to investigate the second possibility, we have tested whether the fraction of tryptophans that had not been quenched by the brominated lipids were accessible to a soluble quencher such as KI.
-Toxin was first allowed to insert into Br-DOPG vesicles in the
absence of KI; the vesicles were then pelleted and resuspended in
buffer (20 mM MES, pH 4.75) containing either 150 mM KI or 150 mM NaCl. Experiments were
performed at 150 mM KI in order to reach maximal quenching
while maintaining the ionic strength at 150 mM (at lower KI
concentrations quenching was lower, data not shown). As illustrated in
Fig. 2B, 48 ± 5% (n = 3) of the residual fluorescence (which had not been quenched by bromines) could
still be quenched by KI, a value that corresponds to quenching of about
19% of the initial fluorescence of
-toxin in solution (compare Fig.
2, A and B). It seems unlikely that higher KI
concentrations would lead to significantly more quenching. Most of the
fluorescence remaining after quenching by both Br-DOPG and KI,
i.e. 21% of the initial fluorescence, is likely due to the
limited efficiency of tryptophan quenching by bromine. Indeed, this
value agrees well with the estimate of Bolen and Holloway (22) that the
quenching efficiency of dibrominated hydrocarbons with bromines located on adjacent carbons is about 80%.
It is noteworthy that the maximum emission wavelength
(max) shifted dramatically upon addition of Br-DOPG
vesicles as well as upon subsequent addition of KI (Fig. 2). Initially
-toxin in solution had a
max of 330.5 nm
corresponding to that of the native toxin (5). After addition of
Br-DOPG vesicles,
max underwent a 9 nm red shift
(
max = 339 nm) indicating that a significant fraction of
the unquenched tryptophan residues were exposed to a hydrophilic
environment. Upon addition of KI,
max shifted back to
lower wavelengths (331.5 nm) indicating that KI had, as expected, quenched solvent-exposed tryptophans and that the remaining fraction that is still fluorescent in the presence of Br-DOPG and KI is in a
hydrophobic environment. This is also what one would expect if the
remaining fluorescent fraction was due to the limited quenching efficiency of bromines.
The preceding analysis of the
accessibility of tryptophan residues to soluble and hydrophobic,
membrane-bound, quenchers indicated that at least 60% of the residues
were accessible to quenching by bromines. Here we have investigated
whether these residues were also accessible to KI. To perform this
experiment we analyzed the effect of KI on the tryptophan fluorescence
of the -toxin channel formed in non-brominated DOPG vesicles.
These experiments first showed that membrane insertion of -toxin led
to an increase in fluorescence intensity as well as a 5 nm red shift in
max (Fig. 3A) indicating that
a fraction of the tryptophan residues became exposed to a hydrophilic
environment upon membrane binding and insertion. The effect of KI was
then analyzed as indicated previously; vesicles containing the
-toxin channel were sedimented by centrifugation and resuspended in
a buffer containing either 150 mM KI or 150 mM
NaCl. As shown in Fig. 3B, only 20 ± 3%
(n = 3) of the fluorescence was quenched by KI, a
percentage that is essentially identical to the one previously obtained
when using Br-DOPG vesicles (Fig. 2). This observation implies that the
large fraction of tryptophans that were quenched by bromine in the
previous experiments were not accessible to KI.
The present observation that the fluorescence increased upon membrane
insertion indicates that the quenching by Br-DOPG is in fact more
pronounced than initially suggested by Fig. 2A. For proper
evaluation of the percentage of quenching, the tryptophan spectrum of
-toxin inserted into Br-DOPG vesicles should be compared with that
of
-toxin inserted into non-brominated DOPG vesicles and not to that
of
-toxin in solution. Taking this correction into account the
percentages of inhibition by Br-DOPG as well as by KI were recalculated
and are shown in Table I. It appears that at least 6 of
the 8 tryptophan residues in
-toxin were accessible to bromine
quenching (taking into account that the quenching efficiency of
bromines is of about 80%).
|
In order to study the
conformation of -toxin when it is bound to the membrane prior to
insertion, we have tested whether we could establish conditions where
most of the toxin would be trapped as a membrane-bound intermediate. We
have previously shown that the channel-forming rate of
-toxin could
be modulated by changing the pH or the amount of negatively charged
lipids in the vesicles (5). Here we have analyzed the effect of
brominated vesicles composed of Br-DOPG/Br-DOPC (50:50) on the
intrinsic fluorescence of
-toxin at various pH values in the hope of
finding conditions where binding would still occur but not insertion. As shown in Fig. 4B, the effect of
Br-DOPG/Br-DOPC vesicles on the intrinsic fluorescence varied
significantly from one pH to another. Under the same conditions, we
have analyzed the
-toxin-induced chloride efflux.
At pH 4.25, the intrinsic fluorescence was readily quenched by Br-DOPG/Br-DOPC in agreement with the fact that channels readily form (5, Fig. 4).
At pH 4.5, the changes in fluorescence observed upon addition of
Br-DOPG/Br-DOPC vesicles were particularly interesting because kinetic
intermediates in the channel formation process could be discriminated.
The fluorescence first increased and then decreased (Fig.
4B). To check whether the decrease was indeed due to bromine quenching, the same experiment was performed with vesicles containing non-brominated DOPG/DOPC (50:50). As shown in the inset of Fig. 4B, the decrease was not observed indicating that the toxin
first bound to the vesicles and probably oligomerized, thereby leading to an increase in fluorescence. A subpopulation of toxin then formed
channels in the vesicles, whereby their tryptophan fluorescence became
quenched by bromines. Under the same experimental conditions, chloride
was rapidly released from the vesicles. The apparent discrepancy
between the observation that most -toxin molecules do not insert
into the membrane and the observation that chloride is rapidly released
is due to the difference in sensitivity between the two methods used.
As mentioned previously, less than two channels are necessary to
release all the chloride from one vesicle within milliseconds (5). In
the present experiments, in order to be able to follow quenching of
tryptophan fluorescence, the equivalent of 80 heptamers of
-toxin
were added per liposome. Therefore, even if only 2% of the
-toxin
molecules inserted into the membrane and formed a channel, chloride
efflux would be observed, whereas the overall tryptophan fluorescence
would not be significantly quenched by bromine. We have previously
shown that at lower protein to vesicles ratios (2 heptamers per
vesicle) chloride is not released at pH 4.5 (5).
It is surprising that such a minute increase in pH (4.25-4.5) led to
such a dramatic drop in the membrane insertion rate. We have previously
described a similarly steep pH dependence for the rate of -toxin
channel formation (5). We have shown that the change in channel
formation rate correlated with the appearance of a molten globule
folding intermediate at the membrane surface (5, 23, 31). Aspartic or
glutamic acids, which have a pKa of around 4 (pKa = 4.4), are possible contributors to this pH
effect. Walker and Bayley (30) have systematically mutated all charged
residues to cysteine in single amino acid mutants and studied the
effect on binding, oligomerization, and channel formation in
erythrocyte membranes. Only three mutations led to a decrease in
hemolytic activity without affecting binding and oligomerization, two
of which were aspartic acids Asp-24 and Asp-152, the latter being
located immediately downstream from the central loop. Therefore
protonation of Asp-152 may promote insertion of the central loop.
At pH 5, addition of Br-DOPG/Br-DOPC vesicles led only to an increase
in fluorescence suggesting that in contrast to what was observed at pH
4.5, most of the toxin underwent a conformational change upon binding
and oligomerization but was trapped at that stage. A minor population,
however, did insert and make channels as witnessed by a chloride efflux
(Fig. 4A). Maximal chloride release was not observed
suggesting that 1% or less of the toxin present in these experiments
formed channels. The intermediate conformation reached by -toxin
when bound to Br-DOPG/Br-DOPC vesicles at pH 5 will be studied in more
detail below.
Finally at pH 7, no change in fluorescence could be observed upon addition of vesicles. Even though the amount of toxin bound to the vesicles was lower at pH 7 than at pH 5 (24 ± 3 instead of 69 ± 2%), the fact that no increase in fluorescence was observed suggests that binding alone is not sufficient to induce the conformational change that leads to an increase in tryptophan fluorescence and that there is an additional pH effect.
Characterization ofThe
conformation of membrane-bound but not inserted -toxin,
i.e.
-toxin bound to DOPG/DOPC vesicles at pH 5, was
studied in more detail. When adding the vesicles to
-toxin about
70% of the protein was bound to the membranes (Fig.
5A). Binding was extremely tight since it
resisted high salt and high pH treatments. SDS-PAGE analysis of the
membrane-associated toxin showed that most of the toxin was in an
oligomeric form (Fig. 7) and therefore corresponded in majority to the
non-lytic pre-pore complex (4, 9, 12).
As illustrated in Fig. 5A, binding led to an increase in fluorescence and a red shift in the maximum emission wavelength as previously observed by Ikigai and Nakae (32, 33). These changes in the tryptophan emission spectrum are likely due to a pH-induced conformational change at the membrane surface and to oligomerization.
The accessibility of tryptophan residues to quenchers was analyzed. As shown in Fig. 5A, addition of Br-DOPG/Br-DOPC vesicles did not lead to a decrease but an increase in fluorescence intensity. The same increase was observed upon addition of non-brominated DOPG/DOPC vesicles (data not shown). Therefore, the tryptophan residues seemed to be inaccessible to quenching by Br-DOPG/Br-DOPC indicating that they must be located at a distance from the plane of the membrane (see "Discussion").
We therefore investigated whether tryptophans were accessible to the
soluble quencher KI. -Toxin was incubated with Br-DOPG/Br-DOPC vesicles for 60 min to allow binding, and the toxin bound to the liposomes was sedimented by centrifugation and resuspended in buffer
(20 mM MES, pH 5) containing either 150 mM KI
or 150 mM NaCl. As shown in Fig. 5B and Table I,
addition of KI led to 78 ± 2% quenching of the tryptophan
fluorescence. The accessibility of the tryptophans to solvent is also
illustrated by the fact that
max was 335.5 nm in the
absence of KI, corresponding to a 5 nm red shift when compared with the
soluble toxin. In contrast in the presence of KI,
max
was 329 nm indicating that KI had quenched the solvent-exposed
tryptophans and that the KI-inaccessible residues were in a very
hydrophobic environment probably in the protein interior or at a
monomer/monomer interface. These observations indicate that most
tryptophans in the membrane-bound
-toxin were accessible to the
soluble quencher in contrast to both the soluble state (KI had no
significant effect on the fluorescence of
-toxin in solution at pH
5) and the membrane-inserted state. This confirms that the toxin
undergoes a first major conformational change upon binding/oligomerization and a second upon insertion/channel
formation.
The
changes in tertiary structure undergone by -toxin upon
oligomerization, membrane binding, and insertion were analyzed by near
UV CD. As shown Fig. 6A,
-toxin at pH 5 has the spectrum of the native soluble toxin characterized by two
negative peaks at 266 and 295 nm and a large positive peak around 280 nm (5, 32). The spectrum of the heptamer was rather similar, but a slight systematic increase in intensity was observed in the 250-280 nm
range indicating a reorganization of the tertiary structure. The
spectrum of the heptamer was similar to the one previously obtained by
Ikigai and Nakae (32) for oligomers prepared in deoxycholate.
The ellipticity measured for -toxin in the presence of DOPG/DOPC
vesicles at pH 5 was more pronounced in the 295-325 nm range when
compared with that of the heptamer in solution, whereas the signal was
somewhat lower in the 280-290 nm range. The signal was mainly due to
the bound toxin since less than 20% (19 ± 3%, n = 5) was unbound as estimated from tryptophan fluorescence measurements (see "Experimental Procedures," under the experimental conditions used for the CD experiments the amount of bound toxin was slightly higher than under the conditions used for the fluorescence
experiments). These observations indicate that binding led to a change
in structure when compared with both the monomer or the oligomer in
solution (Fig. 6A).
Unexpectedly, -toxin inserted into DOPG vesicles (pH 4.75) had a
very weak near UV CD signal (Fig. 6A). The positive
ellipticity observed for the oligomer in the 250-280 nm range gave
rise to a broad negative peak, whereas the well defined peak around 294 nm completely collapsed. The overall weakening of the near UV CD signal
indicated that most of the aromatic residues in the toxin were no
longer in an asymmetric environment and became mobile. A similar
collapse of the near UV CD spectrum was observed upon membrane binding
of the pore-forming toxin colicin A (34).
As shown in the inset of Fig. 6A, the near UV
signal of the -toxin channel increased again upon addition of the
non-ionic detergent Triton X-100. All peaks were, however, shifted to
lower wavelengths (spectrum was taken 5 min after addition of 0.07% detergent) when compared with the peaks of the spectrum of the heptamer
in solution. This observation might explain why Nakae and Ikigai (32)
measured a well defined near UV CD spectrum for oligomeric
-toxin
that had been purified from detergent-solubilized erythrocytes after
treatment with the toxin. These results also indicate that the aromatic
residues in the heptamer have a different conformation in the presence
of a lipid bilayer than in detergent.
The changes in secondary structure occurring upon oligomerization,
membrane binding, and insertion were also analyzed by far UV CD
spectroscopy. As shown in Fig. 6B, the far UV CD spectrum of
-toxin was not significantly affected by heptamerization and membrane binding. The spectra of monomeric
-toxin, heptameric
-toxin in solution, or
-toxin bound to DOPG/DOPC membrane all showed a minimum at 215 nm as previously observed (5, 6, 32). A
significant change in spectrum, however, occurred upon membrane
insertion. The signal became more intense suggesting a slight increase
in the amount of
-sheet, and the minimum was shifted to 217.5 nm.
Proteins containing
-barrels such as porins also show a minimum
ellipticity at 217 nm (35, 36). Therefore this change in far UV CD
spectrum could be due to folding of the central loops into a
-barrel
upon membrane insertion.
In order to further
characterize the changes in conformation undergone by -toxin upon
membrane binding and insertion, we have analyzed the cleavage pattern
of
-toxin after limited proteolysis with Pronase. Treatment of
monomeric
-toxin in solution at pH 5 or 4.75 led to a major band of
approximately 17 kDa (Fig. 7). This pattern is characteristic of
-toxin cleavage in the central loop which leads to two fragments of
similar size (6, 8, 37). Binding of
-toxin to DOPG/DOPC vesicles (pH
5) rendered the protein insensitive to Pronase treatment in agreement
with previous work showing that binding leads to inaccessibility of the
central loop to proteases (3, 4, 6).
Interestingly Pronase treatment of the -toxin channel in DOPG
vesicles (pH 4.75) led to a completely different, very reproducible, cleavage pattern confirming the fact that a conformational change occurred when the bound toxin penetrated into the membrane. A number of
cleavage sites, which remain to be determined, became accessible to the
protease giving rise to eight main fragments with apparent molecular
masses of 28, 25, 20, 16 kDa and 4 bands in the 10-12-kDa range. Given
the sizes of the peptides, cleavage must have occurred all along the
linear sequence of the protein thereby suggesting that the
conformational change that occurred upon insertion was not restricted
to a small region but involved the entire protein.
Channel formation by staphylococcal -toxin is a complex
multi-step process. In order to analyze the configuration of the toxin
at each step, we have established conditions where the toxin accumulated either at the stage of the membrane-bound pre-pore complex
or continued to form the transmembrane channel. We report here that
under certain conditions
-toxin will accumulate at the membrane
surface in the pre-pore state without the need of introducing mutations
that affect channel-forming properties. In the pre-pore state, the
tryptophan residues were shown to be at a distance from the plane of
the membrane and accessible to the soluble quencher KI, whereas they
were inaccessible to KI in the soluble state. These observations
suggest that binding led to a rearrangement of the tryptophan side
chains but that they did not directly interact with the membrane at
this stage (Fig. 8). Upon channel formation, a second conformational
change occurred. Most tryptophan residues reached a hydrophobic
environment where they were accessible to quenching by bromide atoms
attached to the middle of the acyl chain of the lipids suggesting that the C-terminal half of the protein, which contains 7 out of the 8 tryptophans in the protein, came in close contact with the lipid bilayer. Most aromatic residues in the channel were rather mobile since
they no longer gave rise to a CD signal in the near UV. Since it is
unlikely that the C-terminal half of the protein actually penetrates
deeply into the membrane (see "Discussion" below), we speculate
that the tryptophan side chains dip into the membrane in order to
anchor the large extramembranous part of the protein to the lipid
membrane in a manner similar to what has been described for other
membrane proteins such as porins (Fig. 8).
Membrane Binding and Oligomerization
We have shown that at pH
5, -toxin will readily bind to DOPG/DOPC vesicles but will not
insert into the membrane. At the protein concentration used in this
study, most of the membrane-bound toxin was in the heptameric form as
indicated by SDS-PAGE analysis under non-boiling conditions (Fig.
7). The conformational change occurring upon binding and
oligomerization of the toxin led to (i) a relative resistance to
Pronase treatment, (ii) a change in near UV CD spectrum, (iii) an
increase in tryptophan fluorescence, (iv) a red shift in maximum
emission wavelength (
max), (v) accessibility of 78% of
the tryptophan residues to the soluble quencher KI but (vi) total
inaccessibility to quenching by brominated lipids. The first
observation indicates that upon binding/oligomerization the central
loop becomes hidden in agreement with previous observations (3, 4, 6).
The second and third observations show that a rearrangement of the
aromatic residues occurs but that the tertiary structure remains rigid
since the ellipticity in the near UV, although modified, was not
dramatically reduced. The red shift in
max suggests that
a fraction of the tryptophan residues moves to a more hydrophilic
environment, closer to the surface of the protein as also illustrated
by the fact that most tryptophan residues are accessible to KI. The
tryptophan residues, however, remain at a distance from the membrane
plane as they are not accessible to quenching by bromide atoms situated
at the mid-point of the acyl chains. It is not clear what triggers the
conformational changes at the membrane surface. It may be the lower pH
at the interface as previously mentioned (5) and/or the lower
dielectric constant (38).
We have shown that under
conditions where -toxin forms channels, the fluorescence of the
tryptophan residues could be quenched by bromines situated at the
mid-point of the acyl chains. This event, which we have termed
insertion, could not be uncoupled from channel formation and had the
same energy requirement suggesting that these two measurements
correspond to the same event. Insertion leads to a major conformational
change involving all part of the molecule as illustrated by the
appearance of Pronase cleavage sites along the entire primary sequence
(Fig. 7).
The observation that the fluorescence of tryptophans in the -toxin
channel can be quenched by bromide atoms indicates that these residues
come in close proximity of the hydrophobic core of the bilayer. In a
detailed study on the quenching of tryptophan fluorescence by
brominated phospholipids, Bolen and Holloway (22) have shown that
quenching does not necessarily require contact between the chromophore
and the bromide atom, even though the quenching is efficient only at a
very short range. They have estimated that the quenching efficiency is
50% when the tryptophan-bromine pair is separated by a distance of 9 Å. In the light of these observations and considering that in the
present study the bromines were situated approximately 7 Å deep in the
hydrophobic core of the bilayer (39), bromines would not only quench
the fluorescence of tryptophan residues located in the core of the
membrane but also that of tryptophan residues present in the head
group-acyl chain boundary region.
Early studies by electron microscopy indicated that a large proportion
of the -toxin oligomer remains outside the membrane, whereas only a
small portion actually penetrates into the lipid bilayer (40). A large
body of evidence has now confirmed these observations (4, 6, 8, 9, 12,
13, 15, 16). The current view is that only 15 amino acids in the
central loop penetrate deeply into the membrane upon channel formation
(16), although it has not been explicitly shown that other parts of the
protein do not penetrate into the bilayer. It seems unlikely, however,
that most tryptophans that are scattered along the entire protein
(Trp-80, Trp-167, Trp-179, Trp-187, Trp-260, Trp-265, Trp-274, Trp-285)
would all penetrate into the membrane. We rather believe that they dip
into the head group-acyl chain boundary region where they would be
accessible to quenching by bromines at positions 9-10 of the acyl
chain. Other aromatics might also lie at the interface such as the
three tyrosine and one phenylalanine residues that surround the
glycine-rich loop (Tyr-112, Tyr-118, Phe-120, Tyr-148). These aromatic
residues could play a role in anchoring the extramembranous part of the
toxin to the lipid bilayer. Such a role for aromatic residues has been
suggested for certain toxins (41) as well as for a number of membrane
proteins based on the amphipathic nature of these side chains and on
the observation that aromatic residues show a clear preference for the
membrane interface region. The most striking example is that of
bacterial porins in which two rings of aromatic residues surround the
porin trimer at the inner and outer border between the polar and
non-polar parts of the membrane (42, 43). Sequestration of aromatics at
the membrane boundary has also been found in the bacterial photoreaction center (44), cytochrome c oxidase (45), and bacteriorhodopsin (46) although to a lesser extent. A statistical analysis of 115 type I membrane proteins has also shown that tryptophan residues occur preferentially at the extracellular boundary of the
hydrophobic transmembrane segments (47). Finally, it has been suggested
for soluble, membrane-associated proteins such as annexins that
tryptophan residues might contribute to membrane anchoring (48,
49).
The possibility that the tryptophan residues and possibly other
aromatics are dipping into the membrane could partly explain the
weakness of the near UV CD signal of the -toxin channel. A drop in
near UV CD signal indicates that the aromatic residues reach an
isotropic environment and is often interpreted, when studying soluble
proteins, as a partial unfolding of the protein. In the case of
membrane proteins it is, however, not clear how such an observation
should be interpreted. Penetration of aromatics into the membrane
interface might be sufficient to abolish the near UV CD signal without
necessarily evoking partial unfolding of the protein. In the present
case, however, both a partial unfolding (also suggested by the
increased protease sensitivity) and the mobile environment provided by
the lipid head group region are likely to contribute to the low near UV
CD signal. The increase in the far UV CD signal indicates that there is
an increase in
-sheet content which could be due to folding of the
central loops into a 14-stranded
-barrel.
Upon membrane binding and oligomerization,
-toxin undergoes a major conformational change that leads to
rearrangement of tryptophan side chains and their accessibility to
soluble quenchers (Fig. 8). Upon subsequent membrane
penetration, a second change in conformation occurs that is not solely
restricted to the penetration of the central loop into the membrane.
Here we show that tryptophan residues scattered along the entire
C-terminal half of the protein come in close contact with the membrane
and possibly anchor the extramembranous part of the heptameric complex
to the lipid bilayer.
M. Fivaz and J. Gruenberg are greatly acknowledged for their critical reading of the manuscript.
The structure of the -toxin channel at
1.9 Å resolution became available after this work had been accepted
(50). The heptameric complex is a mushroom-shaped structure divided in
three domains: the cap region, the rim regions, and the stem region.
The stem region is thought to span the lipid membrane and is composed
of a 14-stranded
-barrel (residues 110-148 in each promoter).
Between the stem and the rim domains in each protomer is a crevice that is very rich in acidic and aromatic residues and has been suggested to
constitute a lipid binding region. In particular, in agreement with the
present work, this crevice contains several tryptophan residues
(Trp-179, Trp-187, and Trp-260) as well as tyrosine residues (Tyr-112,
Tyr-118, and Tyr-148). Unfortunately the positions of the other five
tryptophans contained in
-toxin are not mentioned. It is important
to note that the structure that has been solved is that of the
detergent-solubilized heptamer, which might be somewhat different from
that of the membrane-inserted heptamer as suggested by the present CD
data.