(Received for publication, August 22, 1994; and in revised form, November 8, 1994)
From the
The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA -endotoxin contains two helices,
5
and
7, that are highly conserved within all different Cry
-endotoxins. To gain information on the mode of action of
-endotoxins, we have used a spectrofluorimetric approach and
characterized the structure, the organization state, and the ability to
self-assemble and to co-assemble within lipid membranes of
5 and
7. Circular dichroism (CD) spectroscopy revealed that
7
adopts a predominantly
-helical structure in methanol, similar to
what has been found for
5, and consistent with its structure in
the intact molecule. The hydrophobic moment of
7 is higher than
that calculated for
5; however,
7 has a lesser ability to
permeate phospholipids as compared to
5. Binding experiments with
7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated
that
7 binds to phospholipid vesicles with a partition coefficient
in the order of 10
M
similar to
5, but with reduced kinetics and in a noncooperative manner, as
opposed to the fast kinetics and cooperativity found with
5.
Resonance energy transfer measurements between fluorescently labeled
pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in
their membrane-bound state,
5 self-associates but
7 does not,
and that
5 coassembles with
7 but not with an unrelated
membrane bound
-helical peptide. Furthermore, resonance energy
transfer experiments, using
5 segments, specifically labeled in
either the N- or C-terminal sides, suggest a parallel organization of
5 monomers within the membranes. Taken together the results are
consistent with an umbrella model suggested for the pore forming
activity of
-endotoxin (Li, J., Caroll, J., and Ellar, D. J.(1991) Nature 353, 815-821), where
5 has transmembrane
localization and may be part of the pore lining segment(s) while
7
may serve as a binding sensor that initiates the binding of the pore
domain to the membrane.
The -endotoxins are produced by Bacillus thuringiensis bacteria during sporulation (for reviews, see
Höfte and Whiteley(1989), Gill et
al.(1992), and Knowles(1994)). These proteins are highly toxic to
insects, and have been used widely as biological insecticides for over
2 decades. The
-endotoxins are hypothesized to insert into the
midgut epithelium of susceptible insect species and to form
transmembrane pores that cause the death of the insects (Knowles and
Ellar, 1987). The toxins are released as protoxins and are then
solubilized in the midgut and activated by gut proteases. The trigger
for the insertion of the pore-forming domain of the toxins into the
epithelium cell membrane is assumed to be a conformational change of
the toxin, which occurs when another domain of the toxin binds to a
receptor presented on the brush-border membranes (Van Rie et
al., 1990; Ahmad and Ellar, 1990).
-Endotoxins bound to
brush-border membrane vesicles (BBMV) (
)cannot be completely
displaced by toxin molecules that bind the same receptor (Hoffmann et al., 1988). This indicates that upon binding a rapid
insertion into the membrane occurs, which may explain the high affinity
of
-endotoxins to BBMV (Li, 1992). Activated
-endotoxins form
single ion channels in planar lipid bilayers (Slatin et al.,
1990; Schwartz et al., 1993) and interact with and permeate
phospholipid vesicles (Yunovitz and Yawetz, 1988; Haider and Ellar,
1989), reflecting pore forming properties of the toxins. That the toxin
can form pores within biological membranes was demonstrated in
patch-clamp experiments with cultured Sf9 insect cells, in which ion
flux occurred upon the addition of
-endotoxin (Schwartz et
al., 1991). Furthermore, the addition of
-endotoxin inhibited
the K
gradient-dependent amino acid transport across
BBMV (Sacchi et al., 1986).
The structure of a
beetle-specific -endotoxin, CryIIIA
endotoxin, has been
determined by x-ray crystallography at 2.5-Å resolution (Li et al., 1991). The toxin is composed of three distinct
domains. Domain I, which includes the N terminus, is composed of a
bundle of six
-helices surrounding a central helix, the
5
helix. Due to the complete shading of
5 by the other helices, it
is not with direct contact with the surrounding solvent molecules.
Domain II is composed of several
-sheets, and domain III has a
-sandwich structure. The structure of domain I of the toxin, the
effect of site-directed mutagenesis in this domain on the activity of
the toxin, and studies with hybrid toxins (Ahmad and Ellar, 1990; Wu
and Aronson, 1992; Ge et al., 1989) suggested that domain I or
part of it is inserted into the membrane and forms a pore. This notion
was further demonstrated by recent studies that showed that a truncated
protein corresponding to domain I of CryIA
-endotoxin
forms ion channels similar to those formed by the intact toxin (Walters et al., 1993). Domain II is thought to be the receptor binding
site, because it contains segments that determine specificity to
insects (Lee et al., 1992; Ge et al., 1989) and
because it contains some of the least conserved (hypervariable)
sequences in the various
-endotoxins (Höfte
and Whiteley, 1989; Knowles and Dow, 1993).
The toxic domain I of
Cry -endotoxin contains two highly conserved regions, Block I,
which predominantly consists of the
5 helix; and Block II, which
contains the
7 helix and part of
6, as well as part of the
conserved
-sheets from the receptor binding domain II.
5 has
been postulated to be a pore-lining segment of Cry
-endotoxin
because it has an amphipathic
-helical structure and because
mutations in this region reduced the toxicity of
-endotoxins
(Ahmad and Ellar, 1990; Wu and Aronson, 1992). Furthermore, a cleavage
site that is important for the activity of CryIVB
-endotoxin was
found in the predicted loop between
5 and
6 helices
(Angsuthanasombat et al., 1993). The location of this cleavage
site, together with its role in larvicidal activity, further strengthen
the hypothesis that
5 plays a major role in the toxic activity of
-endotoxins since a cleaved
5 can interact more efficiently
with the insect membrane.
7 was proposed to be a receptor binding
sensor of
-endotoxin, because it is located in close proximity to
the receptor binding domain II, thus enabling the initiation of the
insertion of the pore-forming domain I into the membrane bilayers (Li et al., 1991).
Recently, peptides corresponding to 5
helices of B. thuringiensis CryIIIA (Gazit and Shai, 1993a)
and CryIA
(Cummings et al., 1994)
-endotoxins have been synthesized and characterized. The
5
peptides interact with phospholipid and insect membranes, permeate
phospholipid vesicles, form ion channels in planar lipid bilayers, and
are cytolytic to insect cells and human erythrocytes. Furthermore,
binding experiments indicated a process whereby several
5 monomers
aggregate within lipid bilayers (Gazit and Shai, 1993a). All of these
observations suggest that
5 segments can serve as structural
elements in the pore formed by
-endotoxins.
In this study, we
utilized a spectrofluorimetric approach to study a possible role of
7 in the toxic mechanism of
-endotoxins, and the organization
of
5 and
7 in their membrane-bound state. The
7 peptide
of B. thuringiensis CryIIIA
endotoxin was synthesized,
fluorescently labeled, and its structure determined in an aprotic
solvent. The peptide was then characterized functionally for its
ability to interact with phospholipid membranes and to permeate them.
Furthermore, resonance energy transfer (RET) measurements were used to
study the organizational state of both
5 and
7 helices within
lipid bilayers. The data reveal that the N terminus of
7 lies on
the surface of the membrane and that the peptide is randomly
distributed within the membrane.
5, on the other hand, tends to
form large aggregates that are presumably organized in a parallel
manner, within phospholipid membranes, and in which the N terminus of
each monomer is located within the hydrophobic core of the membrane.
The data also reveal that membrane-bound
5 can specifically
associate with
7 but not with unrelated membrane-bound amphipathic
-helix. These results strengthen the hypothesis that
5 is a
structural element in the pore formed by
-endotoxins and that
7 has a possible role as a binding sensor of the molecules (Li et al., 1991). Taken together, the results are consistent with
the umbrella model (Li et al., 1991), originally suggested for
the activity of the pore-forming colicin (Lakey et al., 1991).
where []
is the experimentally
observed mean residue ellipticity at 222 nm, and values for
[
]
and
[
]
, corresponding to 0% and 100% helix content at 222 nm, are estimated at 2000 and 30,000
degrees
cm
/dmol, respectively (Chen et al.,
1974; Wu et al., 1981).
The binding isotherms were analyzed as a partition equilibrium (Schwarz et al., 1986, 1987; Rizzo et al., 1987; Beschiaschvili and Seelig, 1990) as described in detail in several other studies (Rapaport and Shai, 1991; Gazti and Shai, 1993a, 1993b), using the following formula:
where X* is defined as the molar ratio of
bound peptide per 60% of the total lipid as had been previously
suggested (Beschiaschvili and Seelig, 1990), K
corresponds to the partition coefficient, and C
represents the equilibrium concentration of free peptide in
solution.
The curve that results from plotting X* versus free peptide, C
, is referred to as the conventional binding
isotherm.
The efficiency of energy transfer (E) was determined by measuring the decrease in the quantum
yield of the donor as a result of the addition of acceptor. E was determined experimentally from the ratio of the fluorescence
intensities of the donor in the presence (I) and
in the absence (I
) of the acceptor at the
donor's emission wavelength, after correcting for membrane light
scattering and the contribution of acceptor emission. The percentage of
transfer efficiency (E) was calculated as
follows:
The correction for light scattering was made by subtracting the signal obtained when unlabeled analogues were added to vesicles containing the donor molecule. Correction for the contribution of acceptor emission was made by subtracting the signal produced by the acceptor-labeled analogue alone.
where I = the initial fluorescence, I
= the total fluorescence observed before
the addition of valinomycin, and I
= the
fluorescence observed after adding the peptide, at time t.
To study the potential role of the 5 and
7 helices
in pore formation by
-endotoxins, peptides and their fluorescently
labeled analogues, with sequences resembling those of the
5 and
7 helical segments of CryIIIA
-endotoxin (residues
193-215 and 259-283, respectively), were synthesized by a
solid phase method and structurally and functionally characterized. Table 1lists the peptides and their designations.
Figure 1:
Kinetics
of binding of 5 and
7 to PS/PC SUVs. NBD-
5 or NBD-
7
(final concentration 0.1 µM) were added to a buffer
solution (50 mM Na
SO
, 25 mM HEPES-sulfate, pH 6.8) containing 420 µM PS/PC
vesicles. Kinetics of binding were followed with time at room
temperature, by measuring the fluorescence emission intensity at 530 nm
with the excitation set at 467 nm. Dashedline,
NBD-
5; dottedline, NBD-
7; continuousline, NBD-Lys-
5. The curve obtained with
NBD-P-
5 is identical to that obtained with NBD-
5 and
therefore is not given.
Figure 2:
Binding isotherm of NBD-7 to
phospholipid vesicles. NBD-
7 binding to PC (squares) and
to PS/PC (circles) vesicles. The binding isotherms were
derived from binding curves by plotting X
* (molar
ratio of bound peptide per 60% of lipid) versusC
(equilibrium concentration of free peptide
in the solution).
Figure 3:
Fluorescence energy transfer dependence on
Rho peptide (acceptors) concentrations using PS/PC vesicles. The
spectra of NBD-Lys-5 (panelA) or NBD-
5 (panelB), the donor peptides (0.05 µM),
were determined in the presence or absence of various concentrations of
the acceptor peptide, Rho-
5. Each spectrum was recorded in the
presence of PC/PS vesicles (100 µM) in 50 mM Na
SO
, 25 mM HEPES-sulfate, at pH
6.8. Solidline, donor alone; dashedline, donor with 0.020 µM acceptor; dottedline, donor with 0.033 µM acceptor; dasheddottedline, donor
with 0.047 µM acceptor.
Figure 4:
Theoretically and experimentally derived
percentage of energy transfer versus bound acceptor/lipid
molar ratio. The amount of lipid-bound acceptor (Rho peptides), C, at various acceptor concentrations was
calculated from the binding isotherms. First, the fractions of bound
acceptor, f
, were calculated for the various
peptide/lipid molar ratios from their binding isotherms. These values
of f
were used to calculate the amount of bound
acceptor, c
(c
=
µM
f
). Filledcircles, NBD-
7 donor, Rho-
7 acceptor; filledsquares, NBD-
5 donor, Rho-cecropinB
acceptor; filledtriangles, NBD-Lys-
5 donor,
Rho-
5 acceptor; opencircles, NBD-
5 donor,
Rho-
5 acceptor; opentriangles, NBD-
5
donor, Rho-
7 acceptor; dashedline, random
distribution of the monomers (Fung and Stryer, 1978), assuming an R
of 51.1 Å (Gazit and Shai,
1993b).
Interestingly the percentage of RET with the
NBD-Lys-5/Rho-
5 pair was significantly lower than that
observed with the NBD-
5/Rho-
5 pair. Since energy transfer is
highly dependent on the distance between the donor and the acceptor
probes (Förster, 1959), this result can be
explained by a situation in which the
5 peptides are organized
within the membrane in a parallel manner.
Figure 5:
Maximal dissipation of the diffusion
potential in vesicles induced by 7. The peptide was added to
isotonic K
free buffer containing SUV,
pre-equilibrated with the fluorescent dye diS-C
-5 and
valinomycin. Maximal fluorescence recovery (when plateau was observed),
measured after mixing the peptides with the vesicles is depicted. Squares, PS/PC vesicles; circles, PC
vesicles.
Figure 6:
Amphiphilic character of 5 and
7
segments. A, Shiffer-Edmundson wheel projections. Shaded
areas indicate hydrophobic residues. B, hydrophobic
moment. The hydrophobic moments were derived using the program DNA
Strider(TM) 1.0. solid line,
5; dashedline,
7.
Although there is vast scientific and commercial interest in
the insecticidal B. thuringiensis -endotoxins, their
precise toxic mechanism has remained elusive. The
-endotoxins are
members of a larger group of membrane pore-forming toxins of bacterial
origin, such as colicin,
-toxin, and aerolysin. All of these
toxins are water-soluble proteins that, in order to exert their
activity, need to undergo a conformational change from hydrophilic to
amphiphilic structures that will allow them to insert into the
hydrophobic core of the cell membrane (Li, 1992). These toxins may
therefore have similar mechanisms for their membrane pore forming
activities (English and Slatin, 1992; Parker and Pattus, 1993).
Domain I of -endotoxins is considered to be the part of the
molecules that causes membrane insertion and pore formation (Li et
al., 1991; Walters et al., 1993). It has also been
speculated that the pores are formed as a result of intermolecular
interactions between
-helices of several toxin monomers of
-endotoxins (Hodgman and Ellar, 1990), which may explain the large
size of the pores (10-20 Å) formed in cell membranes
(Knowles and Ellar, 1987), and the high conductivity of the channels
(
4000 picosiemens) formed in lipid bilayers (Slatin et
al., 1990).
Assuming that the highly conserved structural
elements of Cry -endotoxins play a major role in their toxic
mechanism, we studied the structure, organization, and assembly of the
most conserved segments of
-endotoxins, the
5 and
7
helices. The data reveal that
5 and
7 have different
organizational states; however, they can coassemble in their
membrane-bound state. That
5 is able to self-assemble in its
membrane-bound state, presumably in a parallel manner, was demonstrated
in the following experiments. (i) A high degree of RET between
donor/acceptor-labeled
5 segments was obtained. This value is
higher than that expected for random distribution of monomers ( Fig. 3and Fig. 4). (ii) The degree of RET between
N-terminal donor/N-terminal acceptor labeled pair of
5 is higher
than that obtained with C-terminal donor/N-terminal acceptor labeled
pair of
5 ( Fig. 3and Fig. 4). (iii) The blue shifts
of NBD-labeled
5 analogues are consistent with oriented
localization of the NBD probes, such that the N terminus is embedded
within the hydrophobic core of the membrane, and the C terminus is more
exposed to the solvent (Table 2). (iv) The hydrophobic moment of
5 (Fig. 6B) is quite similar to that calculated
for class M amphipathic
-helices (Segrest et al., 1990).
Class M amphipathic
-helices include lytic peptides that are
hypothesized to penetrate into the hydrophobic core of the membrane and
to form channels or pores (e.g. melittin (Tosteson and
Tosteson, 1981) and pardaxin (Rapaport and Shai, 1992)). Hence, a
bundle of transmembrane amphipathic
-helices (Fig. 6A) can be formed, in which outwardly directed
hydrophobic surfaces interact either with other hydrophobic
transmembrane segments or with the lipid constituents of the membrane,
while the hydrophilic surfaces point inward producing a conducting pore
(Inouye, 1974; Guy and Seetharamulu, 1986; Greenblatt et al.,
1985; Lear et al., 1988). Furthermore, the fact that
5
monomers did not interact with Rho-cecropinB (Fig. 4), which is
an amphipathic
-helical molecule, implies that
5 helices can
specifically interact with each other but not with unrelated membrane
bound amphipathic
-helical molecules. That
5 self-associates
in its membrane-bound state might support the proposal that
-helices from several monomers of
-endotoxins need to
assemble in order to form a pore (Hodgman and Ellar, 1990).
5 may
therefore serve as a structural unit that assists in the
oligomerization of the
-endotoxins monomers. We found that
P-
5 also self-associates in its membrane-bound state (see
``Results''; data not shown). Since P-
5 could not form
large aggregates as indicated in the shape of its binding isotherms
(Gazit and Shai, 1993a), P-
5 presumably forms bundles that contain
only several monomers. Furthermore, P-
5 has a significantly
reduced level of
-helical structure and a lower membrane
permeating activity as compared to
5 (Gazit and Shai, 1993a).
These findings are in line with the observation that a mutant of
-endotoxin with the same substitution in
5 retains
20-30% of its cytotoxic activity (Ahmad and Ellar, 1990).
The
7 monomers, on the other hand, seem to bind randomly onto the
surface of the membrane. The following data support this notion. (i)
RET measurements, and the linearity of the binding isotherms,
demonstrate that
7 does not self-associate in its membrane-bound
state (Fig. 4). Since
7 monomers contain positive and
negative charges, it is energetically unfavored that they will stay as
monomers inside the hydrophobic core of the membrane. (ii) The
blue-shift of the NBD-labeled
7 (Table 2) suggests surface
localization of the N terminus of the peptide. (iii) Although
7
has partition coefficients (Fig. 2, A and B)
similar to those calculated for
5 and other membrane pore-forming
toxins, it has a low membrane permeating activity (Fig. 5). This
low potential to perturb the membranes is probably due to its inability
to penetrate deeply into the hydrophobic core of the membrane. (iv)
7 is not an amphipathic
-helix (Fig. 6A) and
hence does not have the structure hypothesized to be required to form a
bundle of transmembrane amphipathic helices. However, although it seems
to be localized on the surface of the membrane,
7 is protected
from proteolytic digestion by proteinase K (data not shown), similar to
what has been shown with
5 (Gazit and Shai, 1993a). Similar
resistance to proteolytic digestion was found previously with the
antibacterial peptide dermaseptin, which has been shown to bind
randomly on the surface of the membrane (Pouny et al., 1992).
The specificity of the -endotoxins to specific insects is
assumed to be governed by specific receptors that are presented in the
brush border membranes of the midgut (Van Rie et al., 1990).
Binding to receptors triggers conformational changes in the helical
bundle of domain I, thus exposing one or more helices to interact with
and to insert into the midgut membranes. The x-ray structural data
support the notion that
7 may serve as the first helix that senses
the membrane, due to its location in the interface between the
pore-forming domain and the receptor binding domain. According to our
finding
7 does not have properties of a pore lining segment, but
it can serve instead as a segment that binds to the surface of the
membrane. Furthermore, the recognition we found between membrane-bound
5 and
7 in the RET experiments (Fig. 4) may be
consistent with the proposed role of
7 as a binding sensor of
-endotoxin. According to this proposal, upon binding of the
-endotoxin molecules to the receptor,
7 undergoes a
positional change and interacts with the brush border membrane. The
high value of the hydrophobic moment of
7 (Fig. 6B) also support the role of
7 as an
efficient surfactant segment. Due to the affinity of
7 to
5,
the latter also moves out of the
-helical bundle toward the lipid
layer. The movement of
5 results in its better exposure to the
membrane into which it can be inserted to serve as a structural element
in the pore that is formed in the midgut membrane. The cleavage site
that was found in the predicted loop between
5 and
6 helices,
and that was found to be important for the activity of CryIVB
-endotoxin (Angsuthanasombat et al., 1993), probably
helps
5 to insert into the membrane. The other helices are
probably less crucial as structural elements in the pore-forming
mechanism of
-endotoxin due to their variability between classes
of
-endotoxins. Thus it may be that the pore is actually built up
from the aggregation of several
-endotoxin monomers as was
proposed for Cyt
-endotoxins (Maddrell et al., 1989). In
these aggregates
5 may serve as a one of the pore lining segments.
However, it should be indicated that in the crystal structure, which is
not the membrane-bound state of the toxin,
5 is completely
surrounded by the other six helices (Li et al., 1991).
Therefore, the pore-forming domain must undergo a substantial
conformational change to allow
5 to interact with the membrane,
and to serve as a functional element in the transmembrane pore.
The
self-assembly of 5 and the recognition between
5 and
7
found in this study have further general implication. They are in
agreement with increasing evidence that transmembrane helices of
complex membrane proteins can serve as structural units that assist in
the organization and the assembly of the parent proteins. Examples
showing that in vitro assembly of separated transmembrane
helices into functional proteins may occur within a bilayer environment
are: bacteriorhodopsin (Liao et al., 1984; Popot et
al., 1987; Kahn and Engelman, 1992), lactose permease of
Escherichia coli (Bibi and Kaback, 1990; Sahin-Toth and Kaback, 1993),
and the
-adrenergic receptor (Kobilka et al.,
1988). These examples and others (see review by Lemmon and
Engelman(1992)) suggest that transmembrane segments can contribute to
specific recognition and assembly with other proteins as well.
Upon
solving the structure of CryIIIA -endotoxin, and analogous to the
mechanism of pore formation by colicin (Lakey et al., 1991;
Parker and Pattus, 1993), two alternative models for the mode of action
of
-endotoxins have been suggested (Li et al., 1991;
Knowles, 1994): (i) the penknife model, which involves the flip out of
5 and
6 into the membrane like a penknife opening; and (ii)
the umbrella model, which involves the insertion of some segments as a
helical hairpin, while the remaining helices are open on the surface of
the membrane like the ribs of an umbrella. However, no experimental
support is available so far to favor either of the proposed models. Our
data suggesting that
5 can form transmembrane bundles but
7
cannot, and that
7 is probably more exposed to the surface, are
more consistent with the umbrella model (see Fig. 7for a
proposed model modified from Li et al.(1991) and
Knowles(1994)). In this model
7 serves as a binding sensor that
initiates the interaction of the helices of
-endotoxin with
membranes and subsequently binds onto the surface of the membrane. It
stays there like a rib of an umbrella, while
5 serves as a
transmembrane pore-lining segment.
Figure 7:
A schematic presentation of a proposed
model for the interaction of -endotoxin with phospholipid
membranes, modified from Li et al.(1991) and Knowles(1994).
The model shows an intact dimmer and cleaved
5
segments.