©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Assembly and Organization of the 5 and 7 Helices from the Pore-forming Domain of Bacillusthuringiensis -Endotoxin
RELEVANCE TO A FUNCTIONAL MODEL (*)

(Received for publication, August 22, 1994; and in revised form, November 8, 1994)

Ehud Gazit Yechiel Shai(§)

From the Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA -endotoxin contains two helices, alpha5 and alpha7, 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 alpha5 and alpha7. Circular dichroism (CD) spectroscopy revealed that alpha7 adopts a predominantly alpha-helical structure in methanol, similar to what has been found for alpha5, and consistent with its structure in the intact molecule. The hydrophobic moment of alpha7 is higher than that calculated for alpha5; however, alpha7 has a lesser ability to permeate phospholipids as compared to alpha5. Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated that alpha7 binds to phospholipid vesicles with a partition coefficient in the order of 10^4M similar to alpha5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity found with alpha5. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in their membrane-bound state, alpha5 self-associates but alpha7 does not, and that alpha5 coassembles with alpha7 but not with an unrelated membrane bound alpha-helical peptide. Furthermore, resonance energy transfer experiments, using alpha5 segments, specifically labeled in either the N- or C-terminal sides, suggest a parallel organization of alpha5 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 alpha5 has transmembrane localization and may be part of the pore lining segment(s) while alpha7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane.


INTRODUCTION

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) (^1)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 alpha-helices surrounding a central helix, the alpha5 helix. Due to the complete shading of alpha5 by the other helices, it is not with direct contact with the surrounding solvent molecules. Domain II is composed of several beta-sheets, and domain III has a beta-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(c) -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 alpha5 helix; and Block II, which contains the alpha7 helix and part of alpha6, as well as part of the conserved beta-sheets from the receptor binding domain II. alpha5 has been postulated to be a pore-lining segment of Cry -endotoxin because it has an amphipathic alpha-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 alpha5 and alpha6 helices (Angsuthanasombat et al., 1993). The location of this cleavage site, together with its role in larvicidal activity, further strengthen the hypothesis that alpha5 plays a major role in the toxic activity of -endotoxins since a cleaved alpha5 can interact more efficiently with the insect membrane. alpha7 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 alpha5 helices of B. thuringiensis CryIIIA (Gazit and Shai, 1993a) and CryIA (Cummings et al., 1994) -endotoxins have been synthesized and characterized. The alpha5 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 alpha5 monomers aggregate within lipid bilayers (Gazit and Shai, 1993a). All of these observations suggest that alpha5 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 alpha7 in the toxic mechanism of -endotoxins, and the organization of alpha5 and alpha7 in their membrane-bound state. The alpha7 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 alpha5 and alpha7 helices within lipid bilayers. The data reveal that the N terminus of alpha7 lies on the surface of the membrane and that the peptide is randomly distributed within the membrane. alpha5, 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 alpha5 can specifically associate with alpha7 but not with unrelated membrane-bound amphipathic alpha-helix. These results strengthen the hypothesis that alpha5 is a structural element in the pore formed by -endotoxins and that alpha7 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).


EXPERIMENTAL PROCEDURES

Materials

Boc-Gly(phenylacetamido)methyl, Boc-Phe-(phenylacetamido)methyl resins, and dimethylformamide (peptide synthesis grade) were purchased from Applied Biosystems (Foster City, CA), and Boc amino acids were obtained from Peninsula Laboratories. Other reagents for peptide synthesis included trifluoroacetic acid (Sigma), N,N-diisopropylethylamine (Aldrich, distilled over ninhydrin), dicyclohexylcarbodiimide (Fluka), and 1-hydroxybenzotriazole (Pierce). Egg phosphatidylcholine (PC) and phosphatidylserine (PS) from bovine spinal cord (sodium salt, grade I), were purchased from Lipid Products (South Nutfield, United Kingdom). Cholesterol (extra pure) was supplied by Merck (Darmstadt, Germany) and recrystallized twice from ethanol. 3,3`-Diethylthiodicarbocyanine iodide (diS-C(2)-5), NBD-F (4-fluoro-7-nitrobenz-2-oxa-1,3-diazole), and tetramethylrhodamine were obtained from Molecular Probes (Eugene, OR). All other reagents were of analytical grade. Buffers were prepared in double glass-distilled water.

Peptide Synthesis, Fluorescent Labeling, and Purification

The alpha5 and alpha7 peptides were synthesized by the solid phase method on phenylacetamidomethyl resins (0.15 meq) (Merrifield et al., 1982; Gazit and Shai, 1993a). The synthesized peptides were purified by RP-HPLC on a C(4) reversed-phase Vydac column (300-Å pore size). The column was eluted in 40 min, at a flow rate of 0.6 ml/min, using a linear gradient of 25-80% acetonitrile in water in the presence of 0.1% trifluoroacetic acid (v/v). The purified peptides were shown to be homogeneous (99%) by analytical HPLC. The peptides were subjected to amino acid analysis in order to confirm their composition. Labeling of the N terminus of alpha5 and alpha7 peptides was achieved as described previously (Rapaport and Shai, 1991). The labeling of alpha5 with NBD at the epsilon amine of the lysine was performed under similar conditions. However, in that case an N-terminal acetylated peptide, that had been cleaved from the resin, was used. The synthetic peptides were purified to 99% using RP-HPLC as described in the previous section.

Preparation of Vesicles

Small unilamellar vesicles (SUV) were prepared by sonication of PC or PS/PC (1:1 w/w) mixture as described in detail elsewhere (Shai et al., 1990, 1991; Gazit and Shai, 1993a). Vesicles were visualized by using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan) as follows. A drop of vesicles was deposited on a carbon-coated grid and negatively stained with uranyl acetate. The grids were examined, and the vesicles were shown to be unilamellar with an average diameter of 20-50 nm (Papahadjopoulos and Miller, 1967).

CD Spectroscopy

The CD spectrum of the alpha7 peptide was measured with a Jasco J-500A spectropolarimeter. Spectra were scanned in a capped quartz optical cell with a 0.5-mm path length, at room temperature at wavelengths of 250 to 190-200 nm. Seven scans were taken at a scan rate of 20 nm/min. The peptide was scanned at a concentration of 2.0 times 10M, in methanol. Fractional helicities (Wu et al., 1981; Greenfield and Fasman, 1969) were calculated as follows:

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 degreesbulletcm/dmol, respectively (Chen et al., 1974; Wu et al., 1981).

NBD Fluorescence Measurements and Kinetics of alpha5 and alpha7 Binding to SUV Vesicles

Changes in the fluorescence of NBD-labeled peptides were measured upon their binding to vesicles. NBD-labeled peptide (0.1 µM) was added to 2 ml of buffer containing 120 µl (430 µM) of SUV composed of either PC or PC/PS to establish a lipid:peptide ratio in which most of the peptide was bound to lipid (4300:1). After a 2-min incubation, emission spectra were recorded with a LS-50B Spectrofluorimeter (Perkin-Elmer), with excitation set at 467 nm (5-nm slit). To monitor the kinetics of the peptides binding to the vesicles, the increases in the emission intensities of the NBD-labeled peptides were monitored as a function of time, with the excitation wavelength set at 467 nm, using a 10-nm slit, and the emission monitored at 530 nm, using a 5-nm slit (four to five separate experiments).

Binding Experiments

Binding experiments were conducted as described previously (Rapaport and Shai, 1991). Briefly, SUV were added successively to 0.1 µM peptide at 24 °C. Fluorescence intensity was measured as a function of the lipid:peptide molar ratio on a Perkin-Elmer LS-50B Spectrofluorimeter, with excitation set at 467 nm, using a 10-nm slit, and emission was monitored at 530 nm, using a 5-nm slit, in three separate experiments. In order to account for the background signal contributed by the lipids to any given signal, the readings taken when unlabeled peptide was titrated with lipid vesicles were subtracted from the recordings of fluorescence intensity.

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(b)* 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(p) corresponds to the partition coefficient, and C(f) represents the equilibrium concentration of free peptide in solution.

The curve that results from plotting X(b)* versus free peptide, C(f), is referred to as the conventional binding isotherm.

RET Measurements

Fluorescence spectra were obtained at room temperature in a Perkin-Elmer LS50B spectrofluorimeter, with the excitation monochromator set at 467 nm with a 5-nm slit width. Measurements were performed in a 1-cm path length quartz cuvette in a final reaction volume of 2 ml. In a typical experiment, the desired amount of a donor peptide was added to a dispersion of SUV (prepared as described above) in buffer (50 mM Na(2)SO(4), 25 mM HEPES-sulfate, pH 6.8), followed by the addition of acceptor peptide either as a single dose or in several sequential doses. Fluorescence spectra were obtained before and after the addition of the acceptor. Any changes in the fluorescence intensity of the donor due to processes other than energy transfer to the acceptor were determined by substituting unlabeled peptide in place of the acceptor.

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(d)) 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.

Calculation of Hydrophobic Moment

Hydrophobic moments (Eisenberg et al., 1982) were calculated using the DNA Strider(TM) 1.0 program on a Macintosh computer. The program was designed and written by Christian Marck, Service de Biochime, Department de Biologie, Institut de Recherch Fondamental, Commissariat a l'Energie Atomic, France.

Fluorometric Detection of Membrane Pores

Pore-mediated diffusion potential assays were performed as described previously (Sims et al., 1974; Loew et al., 1983; Shai et al., 1990, 1991). In a typical experiment, 4 µl (28.8 µg) of a liposome suspension, prepared in K buffer (50 mM K(2)SO(4), 25 mM HEPES-sulfate, pH 6.8), were diluted in 1 ml of isotonic K free buffer (50 mM Na(2)SO(4), 25 mM HEPES-sulfate, pH 6.8) in a glass tube, to which the fluorescent, potential-sensitive dye diS-C(2)-5 (M(r) = 492) was then added. A 1-µl sample of a 10M valinomycin solution was added to the suspension in order to slowly create a negative diffusion potential inside the vesicles, leading to a quenching of the dye's fluorescence. Once the fluorescence had stabilized, 10-15 min later, peptides were added. The subsequent dissipation of the diffusion potential, reflected as an increase in fluorescence, was monitored on a Perkin-Elmer LS50B Spectrofluorimeter, with the excitation set at 620 nm and emission monitored at 670 nm, with the gain adjusted to 100%. The percentage of fluorescence recovery, F(t), is defined as shown by ,

where I(0) = the initial fluorescence, I(f) = the total fluorescence observed before the addition of valinomycin, and I(t) = the fluorescence observed after adding the peptide, at time t.


RESULTS

To study the potential role of the alpha5 and alpha7 helices in pore formation by -endotoxins, peptides and their fluorescently labeled analogues, with sequences resembling those of the alpha5 and alpha7 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.



CD Spectroscopy

The extent of the alpha-helical secondary structure of the alpha7 peptide was estimated from its CD spectrum (Greenfield and Fasman, 1969) in an aprotic solvent (methanol). alpha7 exhibits a mean residual ellipticity [] of -19,850 degreesbulletcm^2/dmol (spectrum not shown), corresponding to the relatively high fractional helicity value of 60% (Wu et al., 1981), as was found previously for the alpha5 helix (Gazit and Shai, 1993a), and in agreement with the structure of alpha7 in the crystal form (Li et al., 1991).

Localization of the Environment of the NBD Moiety

The fluorescence of NBD is sensitive to its environment, and therefore the probe has been used for polarity and binding studies (Kenner and Aboderin, 1971; Frey and Tamm, 1990; Rapaport and Shai, 1991). Herein, the fluorescence emission spectra of NBD-alpha7, NBD-alpha5, NBD-P-alpha5, and NBD-Lys-alpha5, and of the control NBD-aminoethanol were monitored in aqueous solutions or in the presence of vesicles composed of either PC or PC/PS at pH 6.8. In these experiments, SUVs were used to minimize differential light scattering effects (Mao and Wallace, 1984), and the lipid/peptide molar ratio was elevated (4300:1) so that the spectral contributions of free peptide would be negligible. All the peptides exhibited fluorescence emission maxima at 546-547 nm in buffer, reflecting a hydrophilic environment for the NBD moiety (Rajarathnam et al., 1989; Rapaport and Shai, 1991). However, upon addition of vesicles (either PC or PC/PS) to the solutions containing the NBD-labeled peptides, blue shifts in the emission maxima (toward 526-533 nm) and increases in the fluorescence intensities of the NBD group were observed for all the peptides. Table 2lists the emission maxima of all the peptides in buffer and in the presence of the two lipid systems used. The changes in the spectra of the NBD-labeled peptides reflect relocation of the NBD group into the hydrophobic environment of the lipid bilayers. The data reveal the following. (i) The N terminus of alpha7 is located on the surface of the membrane ((max) = 533 nm), whereas that of alpha5 is buried within the hydrophobic core of the membrane ((max) = 527 nm) (Chattopadhyay and London, 1987), both independent of the lipid composition used. (ii) The C terminus of alpha5 is more exposed to the solvent than the N terminus. No shift or enhancement in the fluorescence emission spectra was detected when the control NBD-aminoethanol was used (Table 2). It should be indicated that we cannot rule out the possibility that the intermolecular organization of the membrane-bound peptide may also affect the environment of the NBD moiety. However, in this study the NBD probe is attached selectively to the N terminus of an extended alpha-helical peptide. Therefore, the effect of the bulk environment (i.e. the hydrophobic interior and the headgroup charge) is probably stronger than the effect of the side chain groups of the amino acids that follow the N terminus.



Kinetics of alpha5 and alpha7 Binding to SUV Vesicles

The kinetics of the peptides' binding to lipid vesicles were studied by monitoring the increases in the emission intensities of NBD labeled peptides upon the addition of phospholipid vesicles, as a function of time. Fig. 1shows the changes in the fluorescence of 0.1 µM NBD-alpha5, NBD-Lys-alpha5, and alpha7 upon the addition of 420 µM PS/PC phospholipids. The kinetics of membrane binding of NBD-P-alpha5 was identical to that observed with NBD-alpha5 and therefore is not given. The data reveal a faster kinetics in the increase of the fluorescence of NBD for NBD-alpha5 and NBD-P-alpha5 as compared to NBD-alpha7. These results suggest that alpha5 binds to phospholipid vesicles faster than alpha7 does. However, we cannot rule out the contribution of a process in which NBD-alpha7 quickly binds to the vesicles, and then in a second step a conformational change occurs that affects the fluorescence of NBD-alpha7 peptide. Furthermore, although NBD-Lys-alpha binds with fast kinetics to the membranes, its fluorescence then decreases slowly until a new plateau is reached.


Figure 1: Kinetics of binding of alpha5 and alpha7 to PS/PC SUVs. NBD-alpha5 or NBD-alpha7 (final concentration 0.1 µM) were added to a buffer solution (50 mM Na(2)SO(4), 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-alpha5; dottedline, NBD-alpha7; continuousline, NBD-Lys-alpha5. The curve obtained with NBD-P-alpha5 is identical to that obtained with NBD-alpha5 and therefore is not given.



Characterization of Binding Isotherms and Determination of Partition Coefficients

The binding isotherm of NBD-alpha7 was constructed as described previously for NBD-alpha5 (Gazit and Shai, 1993a). First, NBD-alpha7 (0.1 µM) was titrated with PC or PS/PC SUV. Plotting the resulting increases in the fluorescence intensities of the NBD-labeled peptide as a function of lipid/peptide molar ratios yields conventional binding curves (data not shown). When unlabeled alpha7 was titrated with lipids up to the maximal concentration used with NBD-labeled peptide, the fluorescence intensities of the corresponding solutions (after subtracting the contribution of the vesicles) remained unchanged. The curves obtained when X(b)* (molar ratio of bound peptide per 60% of the total lipid) was plotted versusC(f) (the equilibrium concentration of free peptide in the solution), are referred to as the conventional binding isotherms. The experimental binding isotherms of the interactions of NBD-alpha7 with PC and PC/PS SUV are presented in Fig. 2. The surface partition coefficients were estimated by extrapolating the initial slopes of the curves to zero C(f) values. The estimated surface partition coefficients, K(p)*, of NBD-alpha7 were 5.2 times 10^4M and 2.5 times 10^4M with PC and PC/PS vesicles, respectively. These K(p)* values are within the range of those obtained for the NBD-alpha5 peptide (Gazit and Shai, 1993a) and other membrane-permeating bioactive peptides such as melittin and its derivatives (Stankowsky and Schwarz, 1990), the Staphylococcus -toxin (Thiaudière et al., 1991), the antibiotic dermaseptin (Pouny et al., 1992), and pardaxin analogues (Rapaport and Shai, 1991). However, the binding isotherms of NBD-alpha7 are linear, suggesting a simple partition process, and are different from those obtained with NBD-alpha5 (Gazit and Shai, 1993a). The isotherms obtained with the latter displayed an initial ``lag'' especially with PC vesicles, i.e. the curves were initially flat, but rose once a threshold concentration was achieved. This behavior is consistent with a process whereby peptides first incorporate into the membrane and then once inside the membrane aggregate to form a pore (Schwarz et al., 1986, 1987; Rizzo et al., 1987). Thus, we may conclude that alpha5 forms aggregates in its membrane-bound state, but that alpha7 does not.


Figure 2: Binding isotherm of NBD-alpha7 to phospholipid vesicles. NBD-alpha7 binding to PC (squares) and to PS/PC (circles) vesicles. The binding isotherms were derived from binding curves by plotting X(b)* (molar ratio of bound peptide per 60% of lipid) versusC(f) (equilibrium concentration of free peptide in the solution).



Fluorescence Energy Transfer Studies

It has been suggested that the shape of a binding isotherm indicates whether or not a particular peptide binds the membranes in a cooperative manner to form large aggregates. However, binding experiments cannot indicate self-association of a particular polypeptide to form small sized bundles, or hetero-association of several distinct monomers. To evaluate whether such bundles are formed, RET measurements were performed as described previously (Gazit and Shai, 1993b). In these experiments alpha5, P-alpha5, and alpha7 were used either as donors (NBD-alpha5, NBD-P-alpha5, NBD-Lys-alpha5, or NBD-alpha7) or as acceptors (Rho-alpha5, Rho-P-alpha5, or Rho-alpha7). In addition, a Rho-labeled amphipathic alpha-helical peptide, Rho-cecropinB (Gazit et al., 1994), was used as a control peptide. The R(0) value for the NBD/Rho donor/acceptor pair was calculated to be 51 Å (Gazit and Shai, 1993b). Examples of typical profiles of the energy transfer from NBD-Lys-alpha5 or NBD-alpha5, to Rho-alpha5 in the presence of PC/PS phospholipid vesicles, are depicted in Fig. 3(A and B), respectively. When Rho-alpha5, at a final concentration of 0.020 µM, 0.033 µM, and 0.047 µM, was added to a mixture of NBD-alpha5 or NBD-Lys-alpha5 (0.05 µM each) and PC/PS lipid vesicles (100 µM), at the NBD excitation wavelength (467 nm), dose-dependent quenching of the donor emission was observed, consistent with energy transfer (Fig. 3, A and B, for NBD-Lys-alpha5 and NBD-alpha5, respectively). To determine the actual percentage of energy transfer, the amounts of lipid-bound acceptors (Rho peptides, termed bound acceptor) at the various acceptor peptide concentrations were calculated from the binding isotherms of the corresponding NBD-labeled peptides as described previously (Pouny et al., 1992). The curves of the experimentally derived percentage of energy transfer versus the bound acceptor/lipid molar ratio are depicted in Fig. 4. A curve corresponding to random distribution of monomers (Fung and Stryer, 1978), assuming a R(0) (distance at which 50% RET occurs) of 51 Å, which was previously calculated for the NBD/Rho donor/acceptor pair (Gazit and Shai, 1993b), is also depicted. A high percentage of energy transfer was obtained with NBD-alpha5/Rho-alpha5, NBD-alpha5/Rho-Lys-alpha5, and NBD-alpha5/Rho-alpha7. These values are markedly higher than those expected for random distribution of monomers (Fig. 4). However, efficiencies of energy transfer between NBD-alpha5 and Rho-cecropinB are similar to those observed for random distribution (Fig. 4). CecropinB is a membrane-permeating alpha-helical antibacterial peptide (Gazit et al., 1994). That alpha5 monomers do not recognize membrane-bound cecropinB suggests that the interactions between alpha5 and alpha5 or alpha7 are rather specific. Furthermore, efficiencies of energy transfer between NBD-alpha7/Rho-alpha7 are also similar to those observed for random distribution (Fig. 4). Thus, we may conclude that, in their membrane-bound state, alpha5 can self-assemble and coassemble with alpha7, but alpha7 cannot self-assemble.


Figure 3: Fluorescence energy transfer dependence on Rho peptide (acceptors) concentrations using PS/PC vesicles. The spectra of NBD-Lys-alpha5 (panelA) or NBD-alpha5 (panelB), the donor peptides (0.05 µM), were determined in the presence or absence of various concentrations of the acceptor peptide, Rho-alpha5. Each spectrum was recorded in the presence of PC/PS vesicles (100 µM) in 50 mM Na(2)SO(4), 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(b), at various acceptor concentrations was calculated from the binding isotherms. First, the fractions of bound acceptor, f(b), were calculated for the various peptide/lipid molar ratios from their binding isotherms. These values of f(b) were used to calculate the amount of bound acceptor, c(b) (c(b) = µM times f(b)). Filledcircles, NBD-alpha7 donor, Rho-alpha7 acceptor; filledsquares, NBD-alpha5 donor, Rho-cecropinB acceptor; filledtriangles, NBD-Lys-alpha5 donor, Rho-alpha5 acceptor; opencircles, NBD-alpha5 donor, Rho-alpha5 acceptor; opentriangles, NBD-alpha5 donor, Rho-alpha7 acceptor; dashedline, random distribution of the monomers (Fung and Stryer, 1978), assuming an R(0) of 51.1 Å (Gazit and Shai, 1993b).



Interestingly the percentage of RET with the NBD-Lys-alpha5/Rho-alpha5 pair was significantly lower than that observed with the NBD-alpha5/Rho-alpha5 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 alpha5 peptides are organized within the membrane in a parallel manner.

Membrane Permeability Induced by the Peptides

alpha7 and its fluorescent derivatives were examined for their efficacy in perturbing the lipid packing and causing leakage of vesicular contents, by utilizing the dissipation of diffusion potential assay. Increasing concentrations of alpha7 or its analogues were mixed either with PC or with PS/PC SUV that had been pretreated with the fluorescent, potential-sensitive dye diS-C(2)-5 and valinomycin. Recovery of fluorescence was monitored as a function of time until a plateau was observed and usually occurred within 10-25 min. Maximal activity of alpha7 and its analogues were plotted versus peptide/lipid molar ratios (Fig. 5). The fluorescently labeled analogues exhibited the same activity as their parent molecules (data not shown). The data reveal that the membrane permeating activity of alpha7 is significantly reduced as compared to alpha5 in both PC and PS/PC vesicles (Gazit and Shai, 1993a). The low permeating activity of alpha7 with PS/PC vesicles may be the result of electrostatic interactions between the positive charges within the peptide and the negative charges of the headgroups of the PS phospholipids. These interactions may lead to some extent of disruption of the lipid packing of the membrane, causing small local leakage as has been suggested for magainin (Matsuzaki et al., 1989).


Figure 5: Maximal dissipation of the diffusion potential in vesicles induced by alpha7. The peptide was added to isotonic K free buffer containing SUV, pre-equilibrated with the fluorescent dye diS-C(2)-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.



Hydrophobic Moments of alpha5 and alpha7

Hydrophobic moments (Eisenberg et al., 1982) of alpha5 and alpha7 were determined (Fig. 6B). Fig. 6B shows that the hydrophobic moment at 100° (which is characteristic of an alpha-helical structure taking 3.6 residues/turn) of alpha7 is significantly higher (40%) than that of alpha5 (1.55 versus 1.11, respectively). However, the hydrophobic moment profile of alpha5 is similar to those of membrane-permeating polypeptides that are assumed to form transmembrane pores, such as melittin or pardaxin (Group M, amphipathic alpha-helices; Segrest et al., 1990).


Figure 6: Amphiphilic character of alpha5 and alpha7 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, alpha5; dashedline, alpha7.




DISCUSSION

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, alpha-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 alpha-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 (geq4000 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 alpha5 and alpha7 helices. The data reveal that alpha5 and alpha7 have different organizational states; however, they can coassemble in their membrane-bound state. That alpha5 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 alpha5 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 alpha5 is higher than that obtained with C-terminal donor/N-terminal acceptor labeled pair of alpha5 ( Fig. 3and Fig. 4). (iii) The blue shifts of NBD-labeled alpha5 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 alpha5 (Fig. 6B) is quite similar to that calculated for class M amphipathic alpha-helices (Segrest et al., 1990). Class M amphipathic alpha-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 alpha-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 alpha5 monomers did not interact with Rho-cecropinB (Fig. 4), which is an amphipathic alpha-helical molecule, implies that alpha5 helices can specifically interact with each other but not with unrelated membrane bound amphipathic alpha-helical molecules. That alpha5 self-associates in its membrane-bound state might support the proposal that alpha-helices from several monomers of -endotoxins need to assemble in order to form a pore (Hodgman and Ellar, 1990). alpha5 may therefore serve as a structural unit that assists in the oligomerization of the -endotoxins monomers. We found that P-alpha5 also self-associates in its membrane-bound state (see ``Results''; data not shown). Since P-alpha5 could not form large aggregates as indicated in the shape of its binding isotherms (Gazit and Shai, 1993a), P-alpha5 presumably forms bundles that contain only several monomers. Furthermore, P-alpha5 has a significantly reduced level of alpha-helical structure and a lower membrane permeating activity as compared to alpha5 (Gazit and Shai, 1993a). These findings are in line with the observation that a mutant of -endotoxin with the same substitution in alpha5 retains 20-30% of its cytotoxic activity (Ahmad and Ellar, 1990).

The alpha7 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 alpha7 does not self-associate in its membrane-bound state (Fig. 4). Since alpha7 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 alpha7 (Table 2) suggests surface localization of the N terminus of the peptide. (iii) Although alpha7 has partition coefficients (Fig. 2, A and B) similar to those calculated for alpha5 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) alpha7 is not an amphipathic alpha-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, alpha7 is protected from proteolytic digestion by proteinase K (data not shown), similar to what has been shown with alpha5 (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 alpha7 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 alpha7 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 alpha5 and alpha7 in the RET experiments (Fig. 4) may be consistent with the proposed role of alpha7 as a binding sensor of -endotoxin. According to this proposal, upon binding of the -endotoxin molecules to the receptor, alpha7 undergoes a positional change and interacts with the brush border membrane. The high value of the hydrophobic moment of alpha7 (Fig. 6B) also support the role of alpha7 as an efficient surfactant segment. Due to the affinity of alpha7 to alpha5, the latter also moves out of the alpha-helical bundle toward the lipid layer. The movement of alpha5 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 alpha5 and alpha6 helices, and that was found to be important for the activity of CryIVB -endotoxin (Angsuthanasombat et al., 1993), probably helps alpha5 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 alpha5 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, alpha5 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 alpha5 to interact with the membrane, and to serve as a functional element in the transmembrane pore.

The self-assembly of alpha5 and the recognition between alpha5 and alpha7 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 beta(2)-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 alpha5 and alpha6 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 alpha5 can form transmembrane bundles but alpha7 cannot, and that alpha7 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 alpha7 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 alpha5 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 alpha5 segments.




FOOTNOTES

*
This research was supported in part by the Israel Cancer Association and the Basic Research Foundation administered by the Israel Academy of Sciences and Humanities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Incumbent of the Adolpho and Evelyn Blum Career Development Chair in Cancer Research. To whom correspondence should be addressed. Tel.: 972-8-342711; Fax: 972-8-344112; bmshai{at}weizmann.weizmann.ac.il.

(^1)
The abbreviations used are: BBMV, brush border membrane vesicle; Boc, butyloxycarbonyl; diS-C(2)-5, 3,3`-diethylthiadicarbocyanine iodide; NBD, 7-nitrobenz-2-oxa-1,3-diazole-4-yl; PC, egg phosphatidylcholine; PS, phosphatidylserine; Rho, tetramethylrhodamine triethylammonium salt; RP-HPLC, reverse phase high performance liquid chromatography; SUV, small unilamellar vesicle.


ACKNOWLEDGEMENTS

We thank Dr. Y. Marikovsky for help in visualization of the phospholipid vesicles using electron microscopy.


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