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Originally published In Press as doi:10.1074/jbc.M305916200 on August 27, 2003

J. Biol. Chem., Vol. 278, Issue 46, 45216-45223, November 14, 2003
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Pore Formation by Equinatoxin II, a Eukaryotic Protein Toxin, Occurs by Induction of Nonlamellar Lipid Structures*

Gregor Anderluh{ddagger}, Mauro Dalla Serra§, Gabriella Viero§, Graziano Guella¶, Peter Macek{ddagger}, and Gianfranco Menestrina§||

From the {ddagger}Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia, §ITC-CNR, Institute of Biophysics, Section at Trento, Via Sommarive 18, 38050 Povo (Trento) Italy, Laboratory of Bioorganic Chemistry, Department of Physics, University of Trento, Via Sommarive 14, 38050 Povo (Trento), Italy

Received for publication, June 5, 2003 , and in revised form, August 26, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pore formation in the target cell membranes is a common mechanism used by many toxins in order to kill cells. Among various described mechanisms, a toroidal pore concept was described recently in the course of action of small antimicrobial peptides. Here we provide evidence that such mechanism may be used also by larger toxins. Membrane-destabilizing effects of equinatoxin II, a sea anemone cytolysin, were studied by various biophysical techniques. 31P NMR showed an occurrence of an isotropic component when toxin was added to multilamellar vesicles and heated. This component was not observed with melittin, {alpha}-staphylococcal toxin, or myoglobin. It does not originate from isolated small lipid structures, since the size of the vesicles after the experiment was similar to the control without toxin. Electron microscopy shows occurrence of a honeycomb structure, previously observed only for some particular lipid mixtures. The analysis of FTIR spectra of the equinatoxin II-lipid complex showed lipid disordering that is consistent with isotropic component observed in NMR. Finally, the cation selectivity of the toxin-induced pores increased in the presence of negatively charged phosphatidic acid, indicating the presence of lipids in the conductive channel. The results are compatible with the toroidal pore concept that might be a general mechanism of pore formation for various membrane-interacting proteins or peptides.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteins and peptides with the capacity to increase membrane permeability have been elaborated by a large number of organisms and are used as toxins, effectors in immune response or apoptosis. One of the most commonly adopted mechanisms is the formation of pores in the targeted membrane as occurs, for example, with pore-forming toxins (PFT)1 (1, 2). Bacterial PFT, protein molecules of Mr > 30,000, usually follow two strategies; they either form a channel via insertion of a de novo generated transmembrane {beta} barrel (examples are staphylococcal {alpha}-toxin, the cholesterol-dependent cytolysins, and the protective antigen of anthrax toxin), or they insert a bundle of preexisting {alpha}-helices through the membrane (like colicins and crystal {delta}-endotoxins) (3, 4). Smaller molecules, like antimicrobial peptides or peptide toxins with Mr between 1000 and 5000, have developed a wider set of mechanisms (5). In fact, besides the {beta}-barrel (e.g. protegrin) (6) and the {alpha}-helix bundle (e.g. alamethicin and other peptaibols) (7), some alternative strategies were found, which directly modify the bilayer organization of the membrane. They range from a generic destabilization (exemplified by the carpet-like model) (8) to the formation of specific mixed lipid-peptide structures, like the toroidal pore, which was observed with magainin (9) and melittin (10).

Actinoporins are a peculiar class of eukaryotic PFT with intermediate Mr, exclusively found in sea anemones. It is a family of cysteineless proteins with Mr around 18,000–20,000 and a preference for sphingomyelin (SM) (11). They form cation-selective pores with a diameter of ~2 nm on cellular and model membranes (12–14). The three-dimensional structure of the soluble state of one actinoporin, equinatoxin II (EqtII; from the sea anemone Actinia equina) was recently solved by x-ray (15) and NMR (16). The molecule is composed of a hydrophobic {beta}-sandwich core, flanked on the opposite sides by two {alpha}-helices. The first 30 N-terminal residues, which include one helix, are the best candidates for pore formation. This part of the molecule is amphipathic in character, is well conserved in all actinoporins, is clearly similar to some membrane-interacting peptides like melittin and fusogenic viral peptides (17), and is the only portion of the molecule that can change conformation without disrupting the general fold (15). Preliminary evidence of the involvement of the N-terminal part in the formation of the transmembrane channel was obtained by a low resolution cysteine-scanning mutagenesis along the molecule (18) and by N-terminal truncations (19). More recently, a complete cysteine-scanning mutagenesis of the region encompassing residues 10–28 clearly demonstrated that this portion adopts {alpha}-helical configuration in the membrane, that the {alpha}-helix is longer than in the soluble form, that it lies on the water-lipid interface in the membrane-bound state, and that it inserts into the membrane to line the pore interior, forming an angle of around 20° with the bilayer normal (20). The slight increase in helicity that occurs upon lipid binding was already noted by FTIR spectroscopy (21) and is consistent with the length of the homologous, membrane-perturbing, helical peptides.

In our current model, initial binding is provided by a cluster of exposed aromatic residues (which includes Trp-112 and Trp-116) (18, 22, 23), followed by N-terminal helix translocation to the surface of the membrane and its insertion into the lipid bilayer (20, 23). The channel entity is not stable enough to be isolated and analyzed as a single unit. Therefore, the number of monomers appearing in the final pore was deduced from indirect experiments. All evidence, gathered from cross-linking (13), steady state (12), and kinetic (24) partitioning experiments, consistently indicated a tetrameric structure. Notably, however, the pore diameter is too large to be formed by a simple bundle of four helices. One possibility to solve this apparent contradiction is to assume that the pore is partially lined by membrane lipids.

In fact, perturbation of the lamellar lipid structure has been noted with some of the helical peptides similar to the N-terminal helix of actinoporins (e.g. mastoparan (25), staphylococcal {delta}-lysin (26), the HIV-1 viral fusion peptide (27), and some synthetic peptides with an amphipathic {alpha}-helical character (28)). In addition, a direct effect of EqtII on the lipid phase has been recently observed and attributed to the separation of a sphingomyelin-enriched phase and to vesiculation (29). Given its similarity with melittin, we suspected that the N-terminal helix of actinoporins could perturb the membrane by giving rise to the formation of a toroidal protein-lipid pore. Direct functional evidence for such a structure was already provided by the toxin-strengthening effect of lipids that favor the positive curvature appearing at the center of the toroidal pore and by the fact that pore formation induced the mobilization of lipid molecules between the two leaflets of the bilayer (30).

Here we report a new investigation of EqtII effects on membrane order, providing original evidence that it can affect the lipid phase by a peculiar mechanism, previously observed only in some specific lipid mixtures. Such a mechanism, which is consistent with the concept that its biological activity is exerted via the formation of a toroidal protein-lipid pore, represents a novel paradigm of protein-induced membrane destabilization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Toxins, Proteins, and Lipids—Native EqtII was isolated from the sea anemones as described (31). Melittin (65–85% pure by high pressure liquid chromatography) and myoglobin (type II, minimum 90%, from whale muscle) were from Sigma; pure {alpha}-toxin from Staphylococcus aureus was a kind gift of Dr. Hungerer (Behringwerk, Marburg, Germany). Lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-diphytanoyl-sn-glycerophosphocholine (DPhPC), phosphatidic acid (PA), egg phosphatidylcholine (PC), and brain SM, were all obtained from Avanti Polar Lipids (Alabaster, AL).

Lipid Vesicle Preparations—PC, POPC, and cholesterol were dissolved in chloroform, SM in chloroform/ethanol (4:1, v/v). Lipid mixtures were vacuum-dried in a film at the bottom of a round flask and further treated in a speed-vac for 1–2 h. The lipid films were resuspended at room temperature by vigorously vortexing in the appropriate buffer, followed by six cycles of freezing and thawing in liquid nitrogen. Such dispersions of multilamellar vesicles (MLV) were either immediately used as such or were extruded through polycarbonate membranes with pores of diameter 400 or 600 nm to prepare large unilamellar vesicles (LUV) of that size. Alternatively, small unilamellar vesicles (SUV) were prepared by sonicating the MLV on ice for 30 min, followed by gentle sedimentation to remove titanium particles released by the sonotrode. Lipid samples with PFT or myoglobin, were prepared similarly except that the buffer used for lipid hydration contained appropriate amounts of the proteins. For actinoporins, mixture POPC/SM or PC/SM in a molar ratio of 1:1 or 2:1 was used, since these two mixtures are the most sensitive in functional assays for binding and permeabilization (13, 24). For the same reason, we used POPC/cholesterol in a molar ratio of 1:1 with {alpha}-toxin (32).

NMR Spectra—31P NMR spectra of the different lipid and lipid/toxin preparations were recorded at 121.4 MHz on a Varian XL-300 spectrometer. Lipid composition was POPC/SM 2:1 (mol/mol), and lipid concentration was 30 mM in 100 mM NaCl, 30 mM Tris-HCl, 1 mM EDTA, pH 7.0 (NMR buffer). 0.4–0.7 ml of the sample were placed in 5-mm Pyrex NMR tubes.

Two different protocols were used (as indicated). The phase-cycled Hahn echo pulse sequence was used on samples that were supplemented with 10% deuterated water to provide an internal lock frequency control (33). Recycle time was 2 s, 90° pulse width was 30 µs, delay between pulses was 100 µs, sweep width was 38.46 kHz, acquisition time was 0.104 s, and transient number was 2000–10,000. Alternatively, spectra were acquired by the single pulse acquisition technique (60° pulse angle and 0.4-s interpulse time) on samples without deuterated water. 2000–4000 transients were used in this case. In all measurements, 8192 complex data points were acquired and exponentially multiplied with 75 Hz prior to Fourier transformation with continuous wave 1H-decoupling during data acquisition. 31P chemical shifts were measured relative to 0.0 ppm indicated by sonicated unilamellar vesicles. Variable temperatures were obtained with a homemade temperature-control unit that was calibrated on the chemical shift difference between the residual OH and CD2H proton of 99.8% deuterated methanol (temperature accuracy ±0.2 °C). Samples were left 10 min to equilibrate after each change of temperature

Measurement of Vesicle Size by Quasielastic Light Scattering (QLS)— MLV, LUV, and SUV size was determined by QLS (34) at a fixed angle (90°) and room temperature, using a laser particle sizer (Malvern Z-sizer 3) equipped with a 5-milliwatt helium-neon laser. For this assay, lipid samples were diluted in NMR buffer to give similar scattering (from 25 to 250 times, depending on their size). A 64-channel correlator was used capable of particle size estimates in the range of 5–5000 nm. Data were analyzed by the cumulant method using Malvern Application Software. The first cumulant provided the apparent diffusion coefficient of the particles (from which the hydrodynamic radius can be derived by the Stokes-Einstein relation), whereas the second cumulant gave the distribution width (35).

Permeabilization of Lipid Vesicles—Permeabilization was assayed by measuring the leakage of calcein (36). MLV were prepared as above, but in the presence of 80 mM calcein (pH 7.0 with NaOH). The external calcein was removed by spinning through minicolumns (Pierce) loaded with Sephadex G50 medium preequilibrated with NMR buffer. Fluorescence was measured in a spectrofluorimeter (SPEX Fluoromax) using a 1-cm semimicroquartz cuvette with 1 ml of NMR buffer with 1 µM lipids continuously stirred. Lysins were added to vesicles at a lipid/protein (or peptide) molar ratio of 1. Excitation wavelength was set to 485 nm, and emission was set to 520 nm with both slits set to 2 nm. Release of calcein (R), a percentage, was calculated according to Equation 1,

(Eq. 1)
where Fmeas, Finit, and Fmax are the measured, initial, and maximal fluorescence, respectively. Fmax was obtained by the addition of Triton X-100 to 1 mM final concentration. Spontaneous leakage was negligible on this time scale. The experiments were run at room temperature.

Electron Microscopy (EM)—Transmission EM was performed with a Philips CM100 microscope operating at 80 kV. A drop of the sample was deposited on the copper grids coated with formvar film and negatively stained with 1% phosphotungstic acid (Sigma). Samples were dried on air before imaging. Images were taken with a BioScan Camera model 792 (Gatan) with CCD resolution of 1024 x 1024 pixels. For size estimation, samples were imaged after the NMR experiment. The dimensions were estimated by measuring the diameter of vesicles from the EM image. Averages for MLV and MLV with EqtII were obtained after fitting dimensions to Gaussian distribution. Averages for MLV with melittin were calculated from the data, since the distribution was not Gaussian in that case.

Preparation of Vesicles for Infrared Spectroscopy—SUV were prepared as above with POPC/SM (1:1) (5 mg/ml) for EqtII and PC/cholesterol (1:1) (6 mg/ml) for {alpha}-toxin. EqtII was applied at a lipid/protein ratio (L/P) of 100. Unbound toxin, estimated by the residual hemolytic activity, was between 5 and 10% and was not removed. Samples were applied straight to the germanium crystal. {alpha}-Toxin was used at L/P of 200 and incubated at 37 °C for 1 h. Free toxin (~35%, as determined by hemolytic activity) was removed by repeated ultrafiltration through polysulfone filters of 300-kDa cut-off (NMWL; Millipore Corp.) as described (37). All preparations were in 10 mM Hepes, pH 7.0.

FTIR—FTIR spectra were collected in the attenuated total reflection configuration, as described (21), on a Bio-Rad FTS 185 spectrometer with a deuterium triglycine sulfate detector and KBr beam splitter, at a nominal resolution of 0.5 cm–1. Three kinds of spectra were taken: toxin alone, lipid alone (SUV of either PC/SM 1:1 or PC/cholesterol 1:1), or toxin-treated SUV. The samples were spread on a 10-reflection germanium crystal (45° cut), flushed with D2O-saturated nitrogen, and housed in a vertical attenuated total reflection attachment (by Specac). Polarized spectra were collected with a rotating wire grid polarizer (fir grid; Specac), manually positioned either parallel (0°) or perpendicular (90°) to the plane of the internal reflections.

The orientation of a structural element was calculated from the dichroic ratio, R = A0°/A90°, where A0° and A90° are the absorption bands of the functional group of that element in the parallel and perpendicular configuration, respectively (38, 39). The form factor, S, was derived from R using Equation 2 (40),

(Eq. 2)
where {theta} represents the angle between the long axis of the molecule under consideration and the transition moment of the investigated vibration; Ex, Ey, and Ez are the components of the electric field of the evanescent wave in the three directions (the z axis being perpendicular to the plane of the crystal), which we calculated according to Harrick expressions for thick films (40, 41). The following order parameters were calculated: (a) SL, for the lipid chains, using either the symmetric or asymmetric CH2 stretching (bands centered at 2850 and 2920 cm–1, respectively) and {theta} = 90°; (b) Samide I', for the amide I' band (integrated between 1600 and 1700 cm–1) with {theta} = 0° (42); and (c) S{alpha}, for the {alpha}-helix, using the Lorentzian component at 1651 ± 1 cm–1, obtained by deconvoluting and curve-fitting the amide I' band (between 1700 and 1600 cm–1) as previously described (21) and using {theta} = 39° (38). To analyze the amide I' band of the lipid-bound toxin, the spectra were previously corrected by subtracting the contribution of the lipid alone, with a weight that eliminates the stretching band of the phospholipid carboxyl groups at 1738 cm–1.

From the order parameter, we calculated the average tilt angle {gamma} of the molecular axis with respect to the z axis according to Equation 3 (40),

(Eq. 3)
By definition, S represents the fraction of molecules aligned with the axis direction, whereas (1 – S) is the fraction remaining disordered. The mean angle {sigma} of the {alpha}-helix axis with respect to the direction of the lipid chains was recalculated from Equation 4 (40),

(Eq. 4)
where {gamma}L is the angle formed by the lipid chains with the z axis. Finally, the lipid to bound toxin ratio (L/Pb) was calculated from A90° according to Equation 5 (42),

(Eq. 5)
where nres represents the total number of residues in the toxin (179 for EqtII and 293 for {alpha}-toxin).

Determination of Ion Selectivity of the EqtII Channel—Electrical properties of EqtII pores were studied using planar lipid membranes (PLM) exactly as described (20). On a weight basis, PLM were made of 20% SM, (80 – x)% of DPhPC and x% PA, with x ranging from 0 to 20. EqtII was added at nanomolar concentration only to the cis side, where voltage was applied. Reversal voltages were measured in a 10-fold KCl gradient and translated into a permeability ratio P+/P– (where P+ and P– refer to cation and anion permeability, respectively) by the Goldmann-Hodgkin-Katz equation. Initially, both sides (volume of 2 ml) were bathed by symmetrical solution of 10 mM Tris, 100 mM KCl, pH 8.0. Thereafter, the trans side was perfused with 10 volumes of 10 mM Tris, 1 M KCl, pH 8.0.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of EqtII on Lipid Isotropy: NMR—The effects of equinatoxin (EqtII) on lipid membranes were first studied by 31P NMR spectroscopy. Initially, pure lipid samples were examined in order to find the most suitable preparation. Four different kinds of vesicles were compared (SUV, LUV400, LUV600, and MLV), which share the same bilayer organization but have quite different size, as shown by QLS (Table I). Ideally, the lipid preparation should display a pure lamellar phase signal so that any toxin-induced change of lipid organization (e.g. from lamellar to hexagonal or isotropic phases) (43, 44) could be detected. Only MLV approximated this behavior, showing, even at the higher temperature, a clear predominance of the broad asymmetric 31P NMR spectra (with low field shoulder) typical of the lamellar phase over a small isomorphic signal (Fig. 1). All other preparations showed the occurrence of a peak around 0 ppm, suggesting the presence of a population of fast tumbling phospholipid molecules, which was predominant, at least at the higher temperature. Clearly, the amount of isotropic phase was inversely correlated to the size of the vesicles and directly related to the temperature. For these reasons, as in other works (25, 26, 28, 29), we used only MLV in the rest.


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  		TABLE I
Diameter of lipid particles measured by quasielastic light scattering or electron microscopy

Dimensions were estimated at room temperature, after having exposed the vesicles to 55 °C.

 



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  		FIG. 1.
31P NMR spectra of the lipid preparations used in this paper. Various kinds of vesicles composed of POPC/SM 2:1 (mol/mol) were prepared at 30 mM lipid concentration in NMR buffer. Spectra were taken by a Varian XL-300 spectrometer at 121 MHz with the phase-cycled Hahn echo pulse sequence. 2000–10,000 transients were accumulated at either 20 °C (left traces) or 50 °C (right traces). At each temperature, the samples were equilibrated for at least 10 min. The size of the lipid vesicles increases from top to bottom.

 
Spectra of MLVs with EqtII present in the buffer during the lipid hydration are given in Fig. 2. Even at a lipid/EqtII molar ratio 100, practically no change was observed at 20 °C. However, upon heating the mixture up to 55 °C, an isotropic peak, in the region of 0 ppm shift, progressively appeared with temperature and toxin content. The isotropic peak was largely reversible with temperature, and after cooling back to 20 °C only a small amount of isotropic phase remained (not shown). The whole cycle was repeatable. Spectra taken with either of the two protocols were qualitatively the same.



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  		FIG. 2.
Effects of EqtII on 31P NMR spectra of MLV. Temperature dependence of 31P NMR spectra of 30 mM POPC/SM 2:1 (mol/mol) MLV either alone (traces on the left, phase-cycled Hahn echo pulse sequence) or with the addition of EqtII in the hydration buffer at L/P of 100 (traces on the right, single pulse protocol). Spectra in each panel were recorded consecutively, from bottom to top, after equilibrating the sample at the indicated temperature for at least 10 min. 4000–10,000 transients were averaged for each spectrum.

 
To clarify whether the observed effects were peculiar of EqtII, controls were performed with the same lipid system and three different polypeptides: S. aureus {alpha}-toxin (a PFT that opens pores by inserting a transmembrane {beta}-barrel) (45); melittin, a pore-forming peptide with a strong destabilizing capacity (46); and myoglobin, a protein that does not associate with the membrane. Neither myoglobin (not shown) nor {alpha}-toxin induced any shift or isotropic peak (Fig. 3) even at high temperatures and high protein content (the lowest L/P used was 25 for myoglobin and 55 for {alpha}-toxin (i.e. lower than that used for EqtII in Fig. 2)). In the case of melittin, instead, MLV were still in lamellar phase at L/P of 20, but a mixed population appeared at L/P of 10, and, finally, all of the signal became isomorphic at L/P of 5, producing a sharp 0 ppm peak even at 20 °C (Fig. 3). At these concentrations, melittin can act as a detergent and generate small mixed micelles, or discoids, which produce the isotropic signal in the 31P NMR spectrum (46, 47).



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  		FIG. 3.
Effects of other polypeptides on 31P NMR spectra of MLV. 31P NMR spectra of 30 mM POPC/SM 2:1 (mol/mol) MLV with {alpha}-toxin (two upper traces), or melittin (lower traces) at the indicated lipid/protein, or lipid/peptide, ratio (L/P). Spectra were recorded with the single pulse protocol at either 20 °C (left traces) or 55 °C (right traces).

 
The changes in NMR spectra in the presence of EqtII could have two reasons. First, an isotropic signal may arise from physical structures, which, at variance with MLV, allow for complete motional averaging of the chemical shift anisotropy (e.g. smaller vesicles or micellar structures) (see Fig. 1). This might happen if the toxin promotes solubilization of MLV into smaller structures by a detergent-like mechanism similar to melittin (Fig. 3). Alternatively, the presence of relatively high toxin concentrations might directly influence and change the lipid phase by promoting isotropic structures like rhombic, cubic, or inverted micellar (44).

Effects of EqtII on Lipid Morphology: QLS—Toxin effects on the size of the MLV were directly assayed by QLS (Table I). Down to an L/P of 50 (corresponding to a w/w ratio of 2), there was no marked change in size induced by EqtII that could justify the appearance of the isotropic signal in the 31P NMR spectrum. Melittin, instead, at L/P of 5 completely destabilized the MLV, producing structures of size comparable with the SUV. Neither of the two control proteins, the pore-forming {alpha}-toxin or the lipid-inert myoglobin, modified the size of the MLV.

Electron Microscopy—The shape and size of control and toxin-treated vesicles was further checked by transmission EM (Fig. 4). Control MLV appeared as cauliflower-like structures with no details within the vesicles and no penetration of the contrasting dye, confirming that they were sealed structures (Fig. 4A). The presence of melittin during MLV formation considerably affected their size (Fig. 4B and Table I), producing much smaller structures already evident at L/P of 10. The effect of EqtII was, instead, markedly different and peculiar. In fact, whereas the size of the complexes formed in the presence of EqtII was approximately the same as that of pure MLV, a multitude of smaller internal structures became visible inside the MLV (Fig. 4, C and D) after they had been exposed to the higher temperature. Obviously, the toxin affected the organization of the internal lipid leaflets, and the newly formed structures could indeed generate the isotropic peak of the NMR signal, while leaving unaffected the overall size estimated by QLS. No internal structures were present when the samples had not been heated. When such structures are magnified (Fig. 4, E and F), it appears that the inner lipid leaflets are not always closed in concentric onion-like shells but can make abrupt bends and several changes of direction. A similar pattern has been previously observed and described as the "honeycomb structure" (Fig. 4G). It occurs in purely lipidic MLV, when they contain a sufficient amount of lipids with the capacity to form the hexagonal phase (HII), such as, for example, PE. In that case, the MLV, instead of being onion-like (as in Fig. 4G, a), could become compartmentalized (as in Fig. 4G, c) when heated through the gel-to-liquid phase transition (44, 48).



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  		FIG. 4.
EM analysis of morphology changes in toxin-treated MLV at room temperature. A, unmodified MLV composed of POPC/SM 2:1. B, the same kind of MLV but with the addition of melittin at L/P of 10. C–F, same MLV with the addition of EqtII at L/P of 50 and the exposure to 50 °C. Magnification increases from C to F. Calibration bars (µm) are reported. G, mechanism of the honeycomb formation in MLV (reproduced, with permission, from Ref. 44). Raising the temperature, contact points between lamellae (b) can undergo localized fusion, with the formation of lipid particles corresponding to HII phase (c), and induce compartmentalization (d).

 
Leakage Experiments—A different mode of action of EqtII and melittin was also confirmed by permeabilization experiments in which they were added to preformed, peptide-free, POPC/SM 2:1 (mol/mol) MLV loaded with the fluorescent marker calcein (Fig. 5). Melittin (at L/P of 1) induced ~90% of release of calcein, indicating that, even if applied externally, it could perturb almost all of the inner layers. EqtII was, at the same L/P, much less efficient, releasing only less than 20% of calcein. This indicates that EqtII, even at 100 times higher toxin/lipid ratios than those used for the NMR experiments, interacts only with the outermost layers and cannot penetrate deeply through the multilayered MLV structure.



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  		FIG. 5.
Permeabilizing capacity of EqtII and melittin on MLV. The release of calcein from POPC/SM 2:1 (mol/mol) MLV is shown. The concentration of both lipids and lysins (melittin or EqtII) was 1 µM. The measurement was done in a final volume of 1 ml of NMR buffer. The arrows indicate the addition of 1 mM Triton X-100 to obtain the signal corresponding to the maximal release.

 
Effects of EqtII on Lipid Orientation: FTIR—Polarization FTIR spectroscopy of EqtII in lipid vesicles was used to investigate toxin effects on the orientation of the lipid chains. Oriented lipid layers were obtained by careful deposition of SUV onto the flat surface of a germanium crystal. Vibrational spectra in the region from 3000 cm–1 to 2800 cm–1, corresponding to the stretching of the CH2 and CH3 groups (Fig. 6), provided the average orientation of the lipid chains with respect to the normal to the plane of the crystal, which is also the plane of the deposited membranes. The dichroic ratio and the order parameter were estimated, and from this the average tilt angle was derived as in Refs. 21 and 49 (Table II). In the case of the lipid alone, we found 29°, a value that is usual for phospholipid membranes above the phase transition (42, 50, 51). Upon interaction with EqtII, a decrease in order was observed, leading to an average angle of around 33° (Table II). To the contrary, {alpha}-toxin does not increase lipid disorder in PC/cholesterol 1:1 membranes (Table II). Such membranes, although slightly less ordered than PC/SM, had to be used because cholesterol is required for {alpha}-toxin interaction (32). Notably, cholesterol per se does not prevent the formation of the honeycomb structure (48).



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  		FIG. 6.
Polarized infrared-attenuated total reflection spectra of EqtII in PC/SM layers. PC/SM (1:1) SUV with absorbed EqtII were deposited onto the surface of a germanium crystal. FTIR spectra were taken with either parallel (0°, solid lines) or perpendicular polarization (90°, dashed lines). Traces are vertically shifted to improve presentation. Indicated features are C-H stretching (a) O-D stretching (b), C=O stretching (c), amide I' (d), and amide II' (e). In the left inset, the region from 3000 to 2800 cm–1 of pure lipids without EqtII is enlarged. Four absorption bands appear that correspond to the symmetric and antisymmetric stretching of the CH2 (at 2850 cm–1 and 2920 cm–1) and the CH3 groups (at 2872 and 2956 cm–1). In the right inset, the amide I' region of EqtII (after subtraction of the lipid contribution) is enlarged. The best curve fit with Lorentzian components is superimposed as a thin solid line to the 90° polarized dashed trace. The Lorentzian components used are reported with thin dotted lines, and the {alpha}-helix component is evidenced in boldface type.

 


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  		TABLE II
Assignment and dichroic ratio of some IR bands observed in PC/SM vesicles alone and with EqtII

 
Spectra collected with perpendicularly polarized light were also used to evaluate the amount of toxin bound to the lipid in the SUV/toxin samples, via Equation 5. L/P values of 115 ± 15 and 300 ± 20 were obtained for EqtII and {alpha}-toxin, respectively. Compared with the initial value in the mixture, they indicated that ~90 ± 2% of EqtII and 67 ± 5% of {alpha}-toxin were bound, which was consistent with the level of hemolytic activity remaining in solution (see "Experimental Procedures"). At variance with our recent work (20), in which L/P values of >=1000 were chosen in order that most of EqtII bound would be still in the monomeric, nonaggregated form, the L/P used here (~100) warrants that the largest fraction of the bound protein is in the pore state (13).

The polarization of the {alpha}-helix component of the amide I' band of membrane-bound EqtII was calculated. We obtained a dichroic ratio (R) = 2.8 (Table II), from which the average angle {gamma}, formed by the helix axis with the vertical to the plane of the crystal surface, was calculated via Equations 1 and 2 using {theta} = 39° (38). We obtained {gamma} = 36° (Table II). However, if we consider that the lipid chains are also forming an angle around the vertical direction and we use Equation 4 to recalculate the approximate tilting of the {alpha}-helices with respect to the lipid chains, we obtain an angle of around 22°. Noting that slightly different values of {theta} for the EqtII {alpha}-helix have been reported (38), we can say that an upper bound for the tilt angle is 35° (as the lower bound of {theta} is 30°). These results are in excellent agreement with pore conductance experiments that we recently reported, indicating that the average orientation of the amphipathic N-term {alpha}-helix, with respect to the pore axis, is 21° (20).

Lipid Dependence of Pore Selectivity—The possibility that mixed lipid/peptide structures occur during pore formation was tested by measuring the selectivity of the pores when the overall surface charge of the membrane was varied. We found that increasing contents of negatively charged lipids of the membrane (by adding PA) increased also the cation selectivity of the EqtII pore (Fig. 7), suggesting that lipid head groups are indeed exposed into the pore lumen.



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  		FIG. 7.
Selectivity of EqtII channels in membranes of different composition. After channel formation by EqtII in a PLM, a 10-fold gradient of KCl was established, and the reversal voltage (Vrev) was determined by finding the potential necessary to clamp the current to zero. Vrev (which ranged from 32 to 39 mV) was converted into a permeability ratio (P+/P–) by the Goldman-Hodgkin-Katz equation. Means ± S.E. of four or five experiments are presented. PLM were composed of 20% SM, the reported amount of PA, and, for the rest, DPhPC.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We observed that EqtII induces the appearance of an isotropic signal in 31P NMR spectra when mixed toxin-lipid MLV are cycled through higher temperatures (Figs. 2 and 3). Similar results were recently reported by another group (29) and attributed to the capacity of EqtII to induce the formation of an SM-enriched phase and to promote vesiculation. Here we used a wider range of techniques and experimental conditions that allowed us to propose a different model for toxin action.

QLS and EM consistently indicated that EqtII affects the organization of the lipid lamellae within the interior of the vesicles, rather than changing the overall size of the multilamellar vesicles (Table I and Fig. 4). It was important to distinguish these two effects, since both could lead to the isotropic signal. EM pictures were particularly informative, since they suggested that EqtII was able to induce the formation of a honeycomb structure in the MLV (Fig. 4), which is consistent with all of our experimental findings. The honeycomb structure was originally described for MLV containing lipids with a propensity to form the hexagonal phase HII and lipids forming the lamellar phase, heated above their main transition temperature (44, 48). In that case, the MLV can go into a state (state c of Fig. 4G), in which small pieces of HII-like phase are formed at the contact points. These do not represent an extended HII phase, and in fact, in 31P NMR spectra, they do not give rise to a typical HII signal but rather to the superposition of an isomorphic signal onto the lamellar one (44, 48). This is exactly what we have observed with EqtII-containing MLV, even with lipids like PC or SM, which do not have a propensity to form HII phase (43). Actually, in the absence of EqtII, the isomorphic phase was irrelevant even at high temperature.

When such MLV are cooled again, they go back to state b of Fig. 4G, with a reversibility that is almost complete in the case of EqtII-treated MLV. State b is a highly compartmentalized lamellar structure fully compatible with what we have seen by EM and NMR. The presence of EqtII pores on each leaflet ensures high membrane permeability to the negative stain, thus explaining why all internal compartments appear black in the EM. In state b, large lamellae are still present, providing the 31P NMR lamellar signal. The lamellae are not always closed around the single compartments, but they go from one spot to another, frequently changing direction. Such changes are evident in the EM enlargement of Fig. 4F. The overall structure is an agglomerate of compartments retaining, as a whole, a big size even at high temperature, consistent with the QLS indication that MLV are not cleaved into smaller structures, as with melittin. The polygonal shape of these compartments, peculiar for the honeycomb structure, is also evident (Fig. 4, E and F).

Until now, the honeycomb structure was observed only in the presence of lipids with the propensity to form HII phase. Therefore, one important question is how EqtII can induce such a propensity in lipids (PC and SM) that do not have it. A possible explanation comes from a model of actinoporin pore formation that we have recently introduced (30), which assumes that these toxins perturb the lamellar organization of the lipid by favoring the formation of a toroidal lipid pore in the bilayer (9, 52). The essential steps of this model are illustrated in Fig. 8. Upon binding to the lipid bilayer (Fig. 8a), the toxin unfolds its amphipathic N-term {alpha}-helix. This helix would absorb into the lipid layer but remains flat on its surface, with the hydrophobic part embedded in the membrane and the hydrophilic part in contact with the solution (Fig. 8b). Upon aggregation of several monomers, the helices might insert through the membrane and simultaneously reorganize the lipid itself to form a toroidal pore (Fig. 8c). Thereby, a hydrophilic pore lined by protein and partially by lipid is formed. This is confirmed by the fact that the cation selectivity of the pore increases if a negatively charged lipid is present in the membrane (Fig. 7) that attracts cations, increasing their local concentration. Notably, whereas the helix in the pore is almost perpendicular to the plane of the membrane (see FTIR results in Fig. 6), it is still lying at the interface between lipid and water, being in the lumen of the toroidal lipid structure (Fig. 8c).



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  		FIG. 8.
Proposed mechanism of pore formation by EqtII in lipid membranes. a, the toxin binds to the lipid bilayer with its amphipathic N-terminal {alpha}-helix still folded as it is in solution. b, the N-terminal {alpha}-helix unfolds and inserts into the lipid layer, remaining flat on the membrane surface, with the hydrophobic part embedded in the membrane and the hydrophilic part in contact with the solution. c, after aggregation, the helices insert into the membrane and promote lipid reorganization into a toroidal pore.

 
With a higher amount of absorbed protein and with the help of higher temperature, the toxin may lead to compartmentalization as depicted in Fig. 9. Toxin pores, present on the different lamellae, are initially randomly distributed (Fig. 9A), but under appropriate circumstances, they may coordinate at given spots to form a kind of local trans-lamellar aggregate (Fig. 9B). Examples of long range coordination of toroidal pores in MLV structures have been already reported (10). New channel-like structures, extending through the lamellar structure from one lamella to the other, may thus form. These structures can be regarded as loci of generation of nonlamellar structure. In fact, simply by reversing the toxin conformational transition that has led to the formation of the toroidal pore (step b to c of Fig. 8 reversed to b again), it is possible to create a layer of toxin adsorbed inside a cylindrical lipid structure (Fig. 9C), which is essentially identical to the contact HII phase of Fig. 4G (although depicted here in a parallel section). Such formation of small HII-like cylinders, or tethers, may occur simultaneously at many places in the MLV as in the proposed honeycomb model and thus originate the observed 31P NMR isomorphic signal. A further transition of the protein to the inserted form (i.e. form c of Fig. 8) could easily lead to the formation of compartments (Fig. 9D) by going through a phase similar to the inverted micellar intermediate and the stalks (43, 53, 54). Although Fig. 9 exemplifies the events only in a two-dimensional representation, it is easy to conceive that the sequential formation and coalescence of many of these tethers, or stalks, could eventually originate the typical three-dimensional compartmentalization of the honeycomb structure (Fig. 4E). In a similar way, inverted micellar intermediates were observed to coalesce and give rise to extended structures like rod micellar intermediates and line defects in pure membranes (43). A similar mechanism of action has been proposed also for F-actin, which establishes toroidal pores and lipid nanotubes (55), and for Bax-type apoptotic proteins (56).



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  		FIG. 9.
Proposed mechanism of MLV compartmentalization induced by EqtII. A, toxin pores are created at random on the different lamellae. B, at contact points, pores may coordinate to form a tubular channel structure extending through the lamellae. C, by acquiring again conformation b of Fig. 8, the toxin may favor the formation a cylindrical lipid structure identical to the elements forming the HII lipid phase. D, a further transition of the protein to the inserted form (i.e. form c of Fig. 8) can finally lead to the formation of compartments. Note that the protein is always in one of the two possible bound states (b or c of Fig. 8) and that the helix is always lying at the water lipid interface.

 
This model is fully consistent with the FTIR results shown in Fig. 6. One implication of the toroidal toxin-lipid pore is that the aliphatic chains of the lipids in the torus, instead of being oriented perpendicular to the plane of the membrane, assume all orientations between perpendicular and parallel. Our polarization experiments indicate that, upon formation of the pore, a fraction of the lipid becomes disordered, which can be calculated in ~8% by the decrease of the order parameter S (Table II). Using the determined L/P of 115, we can thus estimate that around nine lipid molecules are disordered by each EqtII monomer. We can see that this is indeed consistent with the toroidal model. In fact, if we calculate the number of lipid molecules that could fit into the nonoriented toroidal structure, assuming the thickness of the membrane is 6 nm, the radius of the aqueous pore is 1 nm (14), and the volume of the hemitorus of disordered lipid is V = {pi}a2({pi}c – 4/3a) (where a is the radius of the disk that, rotating, generates the torus and c is the radius of rotation), we get a figure of 50–60. This corresponds well to the total number of lipids disordered by the toxin in each pore, if we remember that one pore is formed by a tetrameric aggregate of the toxin (12, 13, 57).

The alternative explanation that lipid disorder could derive from the effect of the toxin portion protruding outside the bilayer is less likely. In fact, {alpha}-toxin, which forms a {beta}-barrel pore inserted perpendicularly through the lamellar membrane (45) (Fig. 3), does not increase lipid disorder in FTIR analysis (Table II), even if the portion protruding outside the bilayer is larger than that of the actinoporin (seven monomers of ~270 amino acid residues (45) instead of four monomers of ~150 residues (20)). One reason for this difference could be that {alpha}-toxin uses {beta}-structure to create the hole through the membrane, whereas EqtII uses an {alpha}-helix (like magainin, for which the toroidal pore was originally introduced) (9, 58). The {alpha}-helix of EqtII shares structural homology with melittin and fusogenic viral peptides (17). In our conditions, melittin behaved different from EqtII and more like a detergent that produces isomorphic structures (SUVs or bicelles). However, it has been shown that, under appropriate conditions of lipid composition, L/P, and temperature, also melittin can induce toroidal pores (10). In addition, many other related {alpha}-helical peptides, including fusogenic viral ones, have a similar lipid-perturbing activity (26–28, 59).


   FOOTNOTES
 
* This work was supported by grants from Consiglio Nazionale delle Ricerche, Ministero Italiano Università e Ricerca Scientifica, and Istituto Trentino di Cultura (to G. A., M. D. S., G.V., G. G., and G. M.) and the Ministry of Education, Science, and Sport of Slovenia (to G. A. and P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 39-0461-314-256; Fax: 39-0461-810-628; E-mail: menes{at}itc.it.

1 The abbreviations used are: PFT, pore-forming toxin(s); DPhPC, 1,2-diphytanoyl-sn-glycerophosphocholine; EqtII, equinatoxin II; EM, electron microscopy; FTIR spectroscopy, Fourier-transformed infrared spectroscopy; LUV, large unilamellar vesicles; L/P, lipid/protein ratio; MLV, multilamellar vesicles; PA, phosphatidic acid; PC, phosphatidylcholine; PLM, planar lipid membranes; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; QLS, quasielastic light scattering; SM, sphingomyelin; SUV, small unilamellar vesicles. Back


   ACKNOWLEDGMENTS
 
We thank Luka Malensek for assistance with the electron microscope. We are also indebted to Alekos Athanasiadis, who first suggested that we investigate the effects of EqtII on the phase morphology of lipids in vesicles.



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