Interaction of the eukaryotic pore-forming cytolysin equinatoxin II with model membranes: 19F NMR studies

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.     

Differential interaction of equinatoxin II with model membranes in response to lipid composition

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (approximately 26 mN m(-1)) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (approximately 33 mN m(-1)), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101-120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.     

Interaction of genistein benzyl derivatives with lipid bilayers—fluorescence spectroscopic and calorimetric study

The purpose of the present paper was to assess the ability of genistein benzyl derivatives to interact with lipid bilayers. Calorimetric and fluorescence spectroscopic measurements revealed that, depending on the details of chemical structure, the studied compounds penetrated bilayers and affected their polar as well as hydrophobic regions. It was also found that physical state of bilayer played some role in flavonoid–lipid interactions.     

Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II

Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or “rafts” in PC membranes, ³¹P and ²H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T₂ relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Effect of lipid on the conformation of the N-terminal region of equinatoxin II: a synchrotron radiation circular dichroism spectroscopic study

Equinatoxin II (EqtII) is a protein toxin that lyses both red blood cells and artificial membranes. Lysis is dependent on the lipid composition, with small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC) and sphingomyelin (SM) (1:1 molar) being lysed more readily than those of phosphatidylcholine alone. Removing the N-terminus of EqtII prevents pore formation, but does not prevent membrane binding. A peptide corresponding to residues 1–32 of EqtII was found using NMR to adopt a helical structure in micelles. To further understand the structural changes that accompany membrane insertion, synchrotron radiation circular dichroism spectra of the N-terminal peptide in a range of model membranes have been analysed. The peptide structure was examined in water, dodecylphosphocholine (DPC) and DPC:SM (5:1) micelles, and SUVs composed of dioleoylphosphatidylcholine (DOPC) or DMPC, together with SM and cholesterol (Chol). The peptide adopted different conformations in different lipids. Although the presence of SM did not affect the conformation in micelles, inclusion of SM in the bilayer-forming lipid increased the helicity of the peptide. This effect was abolished when Chol was added in DOPC but not in DMPC, which may relate to liquid ordered versus disordered phase properties of the lipid. SM may act as a promoter of membrane organisation necessary for membrane lysis by EqtII.     

Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: implications for pore formation

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. The structure of the actinoporin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a beta-sandwich structure formed by two five-stranded beta-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two beta-sheets. 15N relaxation data show uniform backbone dynamics, implying that equinatoxin II in solution is relatively rigid, except at the N terminus; its inferred rotational correlation time is consistent with values for monomeric proteins of similar mass. Backbone amide exchange rate data also support the view of a stable structure, even though equinatoxin II lacks disulfide bonds. As monitored by NMR, it unfolds at around 70 degrees C at pH 5.5. At 25 degrees C the structure is stable over the pH range 2.5-7.3 but below pH 2.5 it undergoes a slow transition to an incompletely unfolded structure resembling a molten globule. Equinatoxin II has two significant patches of positive electrostatic potential formed by surface-exposed Lys and Arg residues, which may assist its interaction with charged regions of the lipid head groups. Tyr and Trp residues on the surface may also contribute by interacting with the carbonyl groups of the acyl chains of target membranes. Data from mutational studies and truncated analogues identify two regions of the protein involved in membrane interactions, the N-terminal helix and the Trp-rich region. Once the protein is anchored, the N-terminal helix may penetrate the membrane, with up to four helices lining the pore, although other mechanisms of pore formation cannot be ruled out.     

Interaction of psychotropic preparations with model lipid membranes

Interaction of psychotropic drugs with model phosphatidylcholine membranes was investigated by fluorescent probes. The data obtained indicate different affinity of the drugs for phosphatidylcholine. The tranquilizers were not bound to the model membranes. The antidepressants were localized in the lipid polar groups area whereas the neuroleptics in the lipid polar groups area and deeper regions of the lipid bilayer.     

Interaction of 3,7-diamino-2,8-dimethyl-5-phenyl phenazinium chloride with model biological membranes and reverse micelles of lipid: a spectroscopic study

The interaction of 3,7-diamino-2,8-dimethyl-5-phenyl phenazinium chloride (Safranine T) with the aqueous as well as reverse micellar solution of a phospholipid 1,2-diacyl-sn-glycero-3-phosphocholine (Azolecithin), a major structural phospholipid in brain, comprising approx 15% of total lipid, primarily localized in grey matter have been studied by absorption and fluorescence spectroscopic studies. The results show the evidence of complex formation of the dye in the ground and in the excited state. The interaction of the dye with the lipid in reverse micellar state is more compared to that in liposomes. An attempt has been made to determine the polarity of the microenvironment of the dye in liposomes or reverse micelles from the spectral studies of the dye in different solvents of known polarity. The polarity functions of the phosphatidylcholine (PC) liposomes are slightly lower compared to that of PC reverse micelles.     

Interaction of the cellular prion protein with raft-like lipid membranes

Conversion of the cellular isoform of the prion protein (PrP(C)) into the disease-associated isoform (PrP(Sc)) plays a key role in the development of prion diseases. Within its cellular pathway, PrP(C) undergoes several posttranslational modifications, i.e., the attachment of two N-linked glycans and a glycosyl phosphatidyl inositol (GPI) anchor, by which it is linked to the plasma membrane on the exterior cell surface. To study the interaction of PrP(C) with model membranes, we purified posttranslationally modified PrP(C) from transgenic Chinese hamster ovary (CHO) cells. The mono-, di- and oligomeric states of PrP(C) free in solution were analyzed by analytical ultracentrifugation. The interaction of PrP(C) with model membranes was studied using both lipid vesicles in solution and lipid bilayers bound to a chip surface. The equilibrium and mechanism of PrP(C) association with the model membranes were analyzed by surface plasmon resonance. Depending on the degree of saturation of binding sites, the concentration of PrP(C) released from the membrane into aqueous solution was estimated at between 10(-9) and 10(-7) M. This corresponds to a free energy of the insertion reaction of -48 kJ/mol. Consequences for the conversion of PrP(C) to PrP(Sc) are discussed.     

Interaction of troponin and tropomyosin: spectroscopic and calorimetric studies

The thermodynamic parameters characterizing the interaction between rabbit fast skeletal muscle troponin and tropomyosin have been determined at 25 degrees C for three solution conditions: buffer containing (A) 1 mM CaCl2, simulating a "turned-on" state, (B) 3 mM MgCl2, simulating a "turned-off" state, and (C) 2 mM ethylenediaminetetraacetic acid, a reference state. The enthalpies were measured in two buffers with different heats of ionization to allow correction for dissociation or uptake of protons. The enthalpies corrected for proton effects are -22.1, -25.4, and -23.5 kcal/mol, respectively, in buffers A, B, and C. The interaction between troponin and tropomyosin in the presence of calcium is accompanied by release of 0.9 mol of proton per mole of complex. Proton effects in the presence of magnesium and in the absence of divalent metal ions were too small to quantitate. The association constants were measured by using tropomyosin labeled with the extrinsic fluorescent probe dansylaziridine, and binding was detected by enhancement of the probe fluorescence. The magnitudes of the association constants for unlabeled troponin are 7.5 X 10(5), 4.2 X 10(5), and 9.5 X 10(5) M-1, respectively, for the three solution conditions corresponding to unitary free energies of -10.4, -10.1, and -10.6 kcal/mol. The unitary entropies for the interaction are -39, -51, and -43 cal/(deg X mol), respectively, for the three solution conditions. Under these conditions, the troponin-tropomyosin interaction is enthalpy driven, and a large unfavorable entropy must be overcome in the formation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes.

Protein K, a recently described outer membrane protein correlated with encapsulation in Escherichia coli (Paakkanen et al., J. Bacteriol. 139:835-841, 1979), has been purified to apparent homogeneity. Purification was based upon the noncovalent association of protein K with peptidoglycan, and the purified protein was shown to form sodium dodecyl sulfate-resistant oligomers on polyacrylamide gels. Incorporation of small amounts (10(-10) to 10(-11) M) of purified protein K into artificial lipid bilayers resulted in an increase, by many orders of magnitude, in membrane conductance. The increased conductance resulted from the formation of large, water-filled, ion-permeable channels exhibiting single-channel conductance in 1.0 M KCl of 1.83 nS. The membrane conductance showed a linear relationship between current and applied voltage and was not voltage induced or regulated. The channel was permeable to large organic ions (e.g., Tris+ Cl-) and, based upon a pore length of 7.5 nm, a minimum channel diameter of 1.2 nm was estimated; these properties resemble values for other enteric porins. The possible biological role of the pores produced by protein K is discussed.     

Interaction of octyl-beta-thioglucopyranoside with lipid membranes.

Octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG) is a nonionic surfactant used for the purification, reconstitution, and crystallization of membrane proteins. The thermodynamic properties of the OTG-membrane partition equilibrium are not known and have been investigated here with high-sensitivity titration calorimetry. The critical concentration for inducing the bilayer <==> micelle transition was determined as cD* = 7.3 mM by 90 degree light scattering. All thermodynamic studies were performed well below this limit. Sonified, unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol were employed in the titration calorimetry experiments, and the temperature was varied between 28 degrees C and 45 degrees C. Depending on the surfactant concentration in the membrane, the partition enthalpy was found to be exothermic or endothermic, leading to unusual titration patterns. A quantitative interpretation of all titration curves was possible with the following model: 1) The partitioning of OTG into the membrane follows a simple partition law, i.e., Xb = Kc(D,f), where Xb denotes the molar amount of detergent bound per mole of lipid and c(D,f) is the detergent concentration in bulk solution. 2) The partition enthalpy for the transfer of OTG from the aqueous phase to the membrane depends linearly on the mole fraction, R, of detergent in the membrane. All calorimetric OTG titration curves can be characterized quantitatively by using a composition-dependent partition enthalpy of the form deltaHD(R) = -0.08 + 1.7 R (kcal/mol) (at 28 degrees C). At low OTG concentrations (R < or = 0.05) the reaction enthalpy is exothermic; it becomes distinctly endothermic as more and more surfactant is incorporated into the membrane. OTG has a partition constant of 240 M(-1) and is more hydrophobic than its oxygen-containing analog, octyl-beta-D-glucopyranoside (OG). Including a third nonionic amphiphile, octa(ethyleneoxide) dodecylether (C12EO8), an empirical relation can be established between the Gibbs energies of membrane partitioning, deltaGp, and micelle formation, deltaGmic, with deltaGp = 1.398 + 0.647 deltaGmic (kcal/mol). The partition constant of OTG is practically independent of temperature and of the cholesterol content of the membrane. In contrast, the partition enthalpy shows a strong temperature dependence. The molar specific heat capacity of the transfer of OTG from the aqueous phase to the membrane is deltaCp = -98 cal/(mol x K). The OTG partition enthalpy is also dependent on the cholesterol content of the membrane. It increases by approximately 1 kcal/mol at 50 mol% cholesterol. As the partition constant remains unchanged, the increase in enthalpy is compensated for by a corresponding increase in entropy, presumably caused by a restructuring of the membrane hydration layer.     

Interaction of insecticides with lipid membranes.

The permeability of liposome membranes is increased by organophosphorus and organochlorinated insecticides at concentrations of 10(-5)--10(-4) M. The order of effectiveness is similar to the toxicity of the compounds to mammals, and is the following for permeation of non-electrolytes and for valinomycin-induced permeation of K+: parathion greater than 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) approximately aldrin greater than malathion greater than lindane. The degree of effectiveness for X-537A-induced permeation of Ca2+ was the following: aldrin greater than or equal to DDT greater than parathion greater than malathion greater than lindane. The organophosphorus compound, ethyl azinphos (10(-4) M), dramatically increases the permeability of liposome membranes to all the tested substances, probably as a consequence of surfactant effects. Some organochlorinated insecticides appear to react with cation ionophores and modulate their motion across lipid membranes. It is suggested that the insecticides may exert some of their toxic actions by modifying certain mechanisms in the cell membrane.     

Membrane insertion of the pore-forming domain of colicin A. A spectroscopic study

In order to gain some insight into the mechanism of insertion into membranes of the pore-forming domain of colicin A and the structure of its membrane-bound form, circular dichroism (in the near and far ultraviolet), fluorescence and ultraviolet spectroscopy experiments were carried out. Because the structure of the water-soluble form of this fragment has been determined by X-ray crystallography, these spectroscopic methods provided valuable information on the secondary structure and the environment of aromatic residues within the two forms of the peptide. These results strongly suggest that the pore-forming domain of colicin A does not undergo drastic unfolding upon insertion into membrane. The conformational change associated with this process is triggered by the negatively charged lipids and probably consists of a reorientation of helix pairs with respect to each other. Exposure of the aromatic residues to the aqueous phase decreases on binding to lipids whilst the exposure of the tryptophans to the membrane phase increases. This cannot occur without a reorientation of helices 3-10. All data from this study support the model presented previously in which the known crystal structure opens like an 'umbrella' inserting the hydrophobic hairpin (helix 8-9) perpendicular to the membrane plane and the helical pair 1-2 and the domain containing the three tryptophans (helices 3-7) lying more or less parallel to the membrane plane. Lipids are bound more tightly to the protein at acidic pH than at neutral pH although a similar lipid protein complex is formed with 1,2-dimyristoyl-sn-glycero(3)-phospho(1)- -sn-glycerol at both pH values.     

A calorimetric and infrared spectroscopic study of the stabilizing solute proline.

We have studied the calorimetric and infrared spectroscopic properties of the amino acid proline which has been implicated in the stabilization of biomacromolecules during reduced water states. It has been suggested that the ability of this molecule to protect biomacromolecules during these stress states may be related to the formation of polymeric aggregates of proline monomers in solution. The structure of this aggregate is thought to be an alternates stack, forming a hydrophilic colloid-like polymer which is thought to interact with hydrophobic moieties of biomacromolecules, reducing the exposed hydrophobic area during reduced water conditions. Calorimetric data presented in this work show that in increasing concentration of proline in solution the enthalpy associated with the melting of bulk water is greatly reduced, indicating strong hydrogen bonding character of proline in aqueous solution. Proline shows two eutectic phase separations at moderate concentrations and one of these eutectics may be the proposed intermolecular state. A partial phase diagram for proline is presented. Fourier-transform infrared spectroscopic data indicate that the COO- asymmetric stretch of proline shows marked splitting with increasing proline concentration. This suggests that the carboxylate is in different environments, with the high energy vibrations representing COO- groups which are participating in the hydrogen bonding pattern associated with the formation of the intermolecular stack. Changes in the CH2 asymmetric and symmetric stretches of the pyrrolidine rings of proline are consistent with the proposed stack structure. We also suggest a possible mechanism by which these intermolecular associations may be important in the protection of biomacromolecules during reduced water states.     

Interaction of KRas4B protein with C6-ceramide containing lipid model membranes

Ras proteins are oncoproteins which play a pivotal role in cellular signaling pathways. All Ras proteins' signaling strongly depends on their correct localization in the cell membrane. Over 30% of cancers are driven by mutant Ras proteins, and KRas4B is the Ras isoform most frequently mutated. C6-ceramide has been shown to inhibit the growth activity of KRas4B mutated cells. However, the mechanism underlying this inhibition remains elusive. Here, we established a heterogeneous model biomembrane containing C6-ceramide. C6-ceramide incorporation does not disrupt the lipid membrane. Addition of KRas4B leads to drastic changes in the lateral membrane organization of the membrane, however. In contrast to the partitioning behavior in other membranes, KRas4B forms small, monodisperse nanoclusters dispersed in a fluid-like environment, in all likelihood induced by some kind of lipid sorting mechanism. Fluorescence cross-correlation data indicate no direct interaction between C6-ceramide and KRas4B, suggesting that KRas4B essentially recruits other lipids. A FRET-based binding assay reveals that the stability of KRas4B proteins inserted into the membrane containing C6-ceramide is reduced. Based on the combined results obtained, we postulate a molecular mechanism for the inhibition of KRas4B mutated cells' activity through C6-ceramide.     

Interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption

The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that anionic lipid membranes can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40s presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40s strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases.