Site-directed mutagenesis at histidines of aerolysin from Aeromonas hydrophila: a lipid planar bilayer study

The role of histidine residues in the formation of channels by the cytolytic toxin aerolysin has been studied in planar lipid bilayers by substituting each of the six histidines in the native protein with asparagine. His341 or His186 mutants had the same channel-forming ability as native toxin, whereas the His332 and His121 mutants were less active. Mutations at His132 and His107, which interfere with the oligomerization of the toxin, drastically reduce pore formation. These findings support the conclusion that oligomerization of the toxin must precede channel formation, and that at least two of the six histidine residues are essential for this to occur. The aerolysin channel is a water-filled pore with an approximate diameter of 9.3 +/- 0.4 A.     

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.     

A rivet model for channel formation by aerolysin-like pore-forming toxins

The bacterial toxin aerolysin kills cells by forming heptameric channels, of unknown structure, in the plasma membrane. Using disulfide trapping and cysteine scanning mutagenesis coupled to thiol-specific labeling on lipid bilayers, we identify a loop that lines the channel. This loop has an alternating pattern of charged and uncharged residues, suggesting that the transmembrane region has a beta-barrel configuration, as observed for Staphylococcal alpha-toxin. Surprisingly, we found that the turn of the beta-hairpin is composed of a stretch of five hydrophobic residues. We show that this hydrophobic turn drives membrane insertion of the developing channel and propose that, once the lipid bilayer has been crossed, it folds back parallel to the plane of the membrane in a rivet-like fashion. This rivet-like conformation was modeled and sequence alignments suggest that such channel riveting may operate for many other pore-forming toxins.     

Site-directed mutagenesis of a single tryptophan near the middle of the channel-forming toxin aerolysin inhibits its transfer across the outer membrane of Aeromonas salmonicida

The channel-forming protein aerolysin must cross both the inner and outer bacterial membranes during its secretion from Aeromonas hydrophila or from Aeromonas salmonicida containing the cloned structural gene. We examined the fate of three mutant proteins in which Trp-227, near the middle of the amino acid chain, was replaced with glycine, leucine, or phenylalanine by site-directed mutagenesis. All three proteins crossed the inner membrane and entered the periplasm in the same way as wild-type, and in each case the signal sequence was removed correctly. Little or none of the proaerolysin substituted with glycine or leucine was released into the culture supernatant. Instead, significant amounts became associated with the outer membrane. The Phe-227 protoxin was secreted by the bacteria but at a reduced rate. The leucine and phenylalanine mutant proteins were purified and compared with native proaerolysin. They were processed correctly to the mature forms by treatment with trypsin, and like native aerolysin, both were resistant to further proteolysis. In each case, processing was followed by the formation of oligomers similar to those produced by native toxin. The hemolytic activity of the processed Phe-227 mutant was one-quarter that of wild-type toxin whereas Leu-227 aerolysin had less than one-hundredth the wild-type activity. These results are further evidence that aerolysin is secreted in at least two steps. As well, they show that the last step, crossing the outer membrane, can be blocked by an apparently small change in the structure of the protein.     

An 8-A projected structure of FhuA, A "ligand-gated" channel of the Escherichia coli outer membrane.

The structure of FhuA, a siderophore and phage receptor in the outer membrane of Escherichia coli, has been investigated by electron crystallography. Bidimensional crystals of hexahistidine-tagged FhuA protein solubilized in N,N-dimethyldodecylamine-N-oxide were produced after detergent removal with polystyrene beads. Frozen-hydrated crystals (unit cell dimensions of a = 124 A, b = 98 A, gamma = 90 degrees ) exhibited a p22121 plane group symmetry. A projection map at 8 A resolution showed the presence of dimeric ring-like structures with an elliptical shape (48 x 40 A). Each monomer was composed of a ring of densities with a radial width of 8-10 A corresponding to a cylinder of beta sheets. Few densities are present inside the barrel, leaving a central channel approximately 25 A in diameter. A projection map of FhuA at 15 A resolution, which was calculated from negatively stained preparations, demonstrated that most of the central channel was masked by extramembrane domains. This map also revealed an asymmetric distribution of extramembrane domains in FhuA, with large domains located mainly on one side of the molecule. Comparison with density maps derived from recent atomic structure allowed further interpretation of the electron microscopy projection structures with regard to long hydrophilic loops governing the selectivity and opening of the channel.     

Channel formation by the glycosylphosphatidylinositol-anchored protein binding toxin aerolysin is not promoted by lipid rafts

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.     

Lipids favoring inverted phase enhance the ability of aerolysin to permeabilize liposome bilayers

Channel formation by the bacterial toxin aerolysin follows oligomerization of the protein to produce heptamers that are capable of inserting into lipid bilayers. How insertion occurs is not understood, not only for aerolysin but also for other proteins that can penetrate membranes. We have studied aerolysin channel formation by measuring dye leakage from large unilamellar egg phosphatidylcholine vesicles containing varying amounts of other lipids. The rate of leakage was enhanced in a dose-dependent manner by the presence of phosphatidylethanolamine, diacylglycerol, cholesterol, or hexadecane, all of which are known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. Phosphatidylethanolamine molecular species with low L-H transition temperatures had the largest effects on aerolysin activity. In contrast, the presence in the egg phosphatidylcholine liposomes of lipids that are known to stabilize the lamellar phase, such as sphingomyelin and saturated phosphatidylcholines, reduced the rate of channel formation, as did the presence of lysophosphatidylcholine, which favors positive membrane curvature. When two different lipids that favor hexagonal phase were present with egg PC in the liposomes, their stimulatory effects were additive. Phosphatidylethanolamine and lysophosphatidylcholine canceled each other's effect on channel formation.     

Crystalline layers and three-dimensional structure of Staphylococcus aureus alpha-toxin

Interaction of the pore-forming protein alpha-toxin from Staphylococcus aureus with lipid components from platelet membranes induces crystal formation of the toxin oligomers. Structure analysis of crystalline areas in either sodium phosphotungstic acid or a sodium phosphotungstic acid/glucose mixture has been performed with electron microscopy and image processing. Ordered domains extending up to a few micrometers were observed, particularly after application of alpha-toxin to pre-formed lipid layers. The crystals, showing tetragonal symmetry, formed either separate two-dimensional sheets or three-dimensional piles of layers. The corresponding unit cell parameter of the single layer was a = b = 109.4 A (standard deviation 2.1 A, n = 21). Incubation of the toxin with intact membranes or extracted lipids as well as application of the lipid layer technique resulted in congruous crystalline properties. The projected averaged alpha-toxin oligomer shows cyclic symmetry with a stain-filled space in the centre. The bulk of the three-dimensional model consists of four asymmetric protein units forming a ring. In addition, a small domain covers the central cavity at the face of the protein opposite to the underlying lipid. The conditions under which the tetragonal arrays are formed on the lipid layers suggest that the alpha-toxin molecule is in a conformation binding to a hydrophobic surface rather than fully inserted into a lipid bilayer.     

Aerolysin,a hemolysin fromAeromonas hydrophila,forms voltage-gated channels in planar lipid bilayers

Summary The cytolytic toxin aerolysin was found to form ion channels which displayed slight anion selectivity in planar lipid bilayers. In voltage-clamp experiments the ion current flowing through the channels was homogeneous indicating a defined conformation and a uniform size. The channels remained open between –70 to +70 mV, but outside this range they underwent voltage-dependent inactivation which was observed as open-closed fluctuations at the single-channel level. Zinc ions not only prevented the formation of channels by inhibiting oligomerization of monomeric aerolysin but they also induced a closure of preformed channels in a voltage-dependent fashion. The results of a Hill plot indicated that 2–3 zinc ions bound to a site within the channel lumen. Proaerolysin, and a mutant of aerolysin in which histidine 132 was replaced by an asparagine, were both unable to oligomerize and neither could form channels. This is evidence that oligomerization is a necessary step in channel formation.     

New structures help the modeling of toxic amyloidbeta ion channels

The mechanism of amyloid toxicity is poorly understood and there are two schools of thought in this hotly debated field: the first favors membrane destabilization by intermediate-to-large amyloid oligomers, with consequent thinning and non-specific ion leakage; the second favors ion-specific permeable channels lined by small amyloid oligomers. Published results currently support both mechanisms. However, the amyloidbeta (Abeta) peptide has recently been shown to form a U-shaped 'beta-strand-turn-beta-strand' structure. This structure and the available physiological data present a challenge for computational biology--to provide candidate models consistent with the experimental data. Modeling based on small Abeta oligomers containing extramembranous N-termini predicts channels with shapes and dimensions consistent with experimentally derived channel structures. These results support the hypothesis that small Abeta oligomers can form ion channels. Molecular dynamics modeling can provide blueprints of 3D structural conformations for many other amyloids whose membrane association is key to their toxicity.     

Role of disulfide bonds in the oligomeric structure and protease resistance of recombinant and native Treponema pallidum surface antigen 4D.

Recombinant Treponema pallidum surface antigen 4D isolated from Escherichia coli formed a protease-resistant ordered ring structure composed of 19,000-dalton subunits. On gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the higher oligomers of recombinant 4D migrated with molecular masses that were nearly multiples of the 190,000-dalton basic ordered ring. Reduction at room temperature with 2-mercaptoethanol converted the 190,000-dalton ordered ring and the higher oligomers to a 160,000-dalton form and the dissociated monomer. A 190,000-dalton form of 4D was identified in sodium dodecyl sulfate-solubilized T. pallidum after reduction at room temperature. Disulfide bonds stabilized both native and recombinant 4D oligomers against dissociation by heating in detergent without a reducing agent. Electron microscopy of recombinant 4D revealed that the characteristic ordered ring structure was maintained after reduction. Reduction of 4D under conditions that preserved the ordered ring structure did not affect the resistance of the molecule to digestion with proteinase K. The properties of 4D suggest that it may fulfill an important structural role in the T. pallidum outer membrane.     

Model for a helical bundle channel based on the high-resolution crystal structure of trichotoxin_A50E

Trichotoxin_A50E is an 18-residue peptaibol antibiotic which forms multimeric transmembrane channels through self-association. The crystal structure of trichotoxin has been determined at a resolution of 0.9 A. The trichotoxin sequence contains nine helix-promoting Aib residues, which contribute to the formation of an entirely helical structure that has a central bend of 8-10 degrees located between residues 10-13. Trichotoxin is the first solved structure of the peptaibol family that is all alpha-helix as opposed to containing part or all 3(10)-helix. Gln residues in positions 6 and 17 produce a polar face, and are proposed to form the channel lumen. An octameric model channel has been constructed from the crystal structure. It has a central pore of approximately 4-5 A radius, a size sufficient to enable transport of ions, with a constricted region at one end, formed by a ring of Gln6 residues. Electrostatic calculations are consistent with it being a cationic channel.     

Two different oligomeric states of the RuvB branch migration motor protein as revealed by electron microscopy

In prokaryotes, the RuvA, B, and C proteins play major roles at the late stage of DNA homologous recombination, where RuvB complexed with RuvA acts as an ATP-dependent motor for branch migration. The oligomeric structures of negatively stained and frozen hydrated RuvB from Thermus thermophilus HB8 were investigated by electron microscopy. RuvB oligomers free of DNA formed a ring structure of about 14 nm in diameter. The averaged top view image clearly indicated a sevenfold symmetry, suggesting that it exists as a heptamer. The RuvB oligomers complexed with duplex DNA formed a smaller ring of about 13 nm in diameter. The averaged top view images represented a sixfold symmetry. This difference in oligomerization indicates that the oligomeric structure of RuvB may convert from a heptamer to a hexamer upon DNA binding. In addition, this finding provides the lesson that great care should be taken in investigating the subunit organizations of DNA binding proteins, because their oligomeric states are more sensitive to DNA interactions than expected.     

Mega assemblages of oligomeric aerolysin-like toxins stabilized by toxin-associating membrane proteins

Most β pore-forming toxins need to be oligomerized via receptors in order to form membrane pores. Though oligomerizing toxins frequently form SDS-resistant oligomers, it was questionable whether SDS-resistant oligomers reflected native functional toxin complexes. In order to elucidate the essence of the cytocidal assemblages, oligomers of aerolysin-like toxins, aerolysin, parasporin-2 and epsilon toxin, were examined with or without SDS. On Blue Native PAGE, each toxin, which had been solubilized from target cells with mild detergent, was a much larger complex (nearly 1 MDa) than the typical SDS-resistant oligomers (～200 kDa). Size exclusion chromatography confirmed the huge toxin complexes. While a portion of the huge complexes were sensitive to proteases, SDS-resistant oligomers resist the proteolysis. Presumably the core toxin complexes remained intact while the cellular proteins were degraded. Moreover, intermediate complexes, which included no SDS-resistant oligomers, could be detected at lower temperatures. This study provides evidence for huge functional complexes of β pore-forming toxins and emphasizes their potential variance in composition.     

Partial purification of the rat erythrocyte receptor for the channel-forming toxin aerolysin and reconstitution into planar lipid bilayers

The cytolytic toxin aerolysin binds to a receptor on the surface of eukaryotic cells. Murine erythrocytes are among the most sensitive to the toxin. Here we describe the detergent solubilization and partial purification of the receptor from rat erythrocytes. We show that it can be successfully incorporated into planar lipid bilayers, greatly decreasing the concentration of aerolysin required to form channels. Exploiting the ability of the receptor to bind aerolysin after SDS electrophoresis and blotting, we obtain evidence that it is a 47 kDa glycoprotein that is sensitive to proteases and N-glycosidase. It may correspond to CHIP28, the water channel of the human erythrocyte.     

Aggregation and porin-like channel activity of a beta sheet peptide

Beta sheet peptides (e.g., amyloid beta) are known to form ion channels in lipid bilayers possibly through aggregation, though the channel structure is not clear. We have recently reported that a short beta sheet peptide, (xSxG)(6), forms porin-like voltage-gated channels in lipid bilayers [Thundimadathil et al. (2005) Biochem. Biophys. Res. Commun. 330, 585-590]. To account for the porin-like activity, oligomerization of the peptide into a beta barrel-like structure was proposed. In this work, peptide aggregation in aqueous and membrane environments and a detailed study of channel properties were performed to gain insight into the mechanism of channel formation. The complex nature of the channel was revealed by kinetic analysis and the occurrence of interconverting multiple conductance states. Ion channels were inhibited by Congo red, suggesting that the peptide aggregates are the active channel species. Peptide aggregation and fibril formation in water were confirmed by electron microscopy (EM) and Congo red binding studies. Furthermore, oligomeric structures in association with lipid bilayers were detected. Circular dichroism of peptide-incorporated liposomes and peptide-lipid binding studies using EM suggest a lipid-induced beta sheet aggregation. Gel electrophoresis of peptide-incorporated liposomes showed dimeric and multimeric structures. Taken together, this work indicates insertion of (xSxG)(6) as oligomers into the lipid bilayer, followed by rearrangement into a beta barrel-like pore structure. A large peptide pore comprising several individual beta sheets or smaller beta sheet aggregates is expected to have a complex behavior in membranes. A dyad repeat sequence and the presence of glycine, serine, and hydrophobic residues in a repeated pattern in this peptide may be providing a favorable condition for the formation of a beta barrel-like structure in lipid bilayers.     

Electrostatics studies and molecular dynamics simulations of a homology model of the Shaker K+ channel pore

A homology model of the pore domain of the Shaker K+ channel has been constructed using a bacterial K+ channel, KcsA, as a template structure. The model is in agreement with mutagenesis and sequence variability data. A number of structural features are conserved between the two channels, including a ring of tryptophan sidechains on the outer surface of the pore domain at the extracellular end of the helix bundle, and rings of acidic sidechains close to the extracellular mouth of the channel. One of these rings, that formed by four Asp447 sidechains at the mouth of the Shaker pore, is shown by pK(A) calculations to be incompletely ionized at neutral pH. The potential energy profile for a K+ ion moved along the central axis of the Shaker pore domain model selectivity filter reveals a shallow well, the depth of which is modulated by the ionization state of the Asp447 ring. This is more consistent with the high cation flux exhibited by the channel in its conductance value of 19 pS.     

Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol,some cholesterol esters,and cholesterol derivatives: a TEM study

The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer approximately 8 nm in outer diameter. Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate. The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces. VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain. At the air/water interface, the VCC oligomers are likely to be selectively oriented with the hydrophobic domain facing the air. Despite some molecular disorder/plasticity within the oligomers, multivariate statistical analysis and rotational self-correlation using IMAGIC-5 strongly suggest the presence of sevenfold rotational symmetry. To correlate the electron microscopy data with on-going biochemical and permeability studies using liposomes of varying lipid composition, the direct interaction of VCC with several cholesterol derivatives and other steroids has been examined. 19-Hydroxycholesterol and 7 beta-hydroxycholesterol both induce VCC oligomerization. beta-Estradiol, which does not possess an aliphatic side chain, also efficiently induces VCC oligomer formation, as does cholesteryl acetate. Cholesteryl stearate and oleate and the C22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol fail to induce VCC oligomerization, but binding of the monomer to the surface of these steroids does occur. Stigmasterol has little tendency to induce oligomer formation, and oligomers are largely confined to the edge of the bilayers; ergosterol has even less oligomerization ability. Attempts to solubilize and stabilize the VCC oligomers from cholesterol suspensions have been pursued using the neutral surfactant octylglucoside. Although individual solubilized oligomers have been defined which exhibit a characteristic cytolysin channel conformation in the side-on orientation, a tendency remains for the oligomers to cluster via their hydrophobic domains.     

Activation of the hole-forming toxin aerolysin by extracellular processing.

A precursor-product relationship between aerolysin and a protein with a higher molecular weight was observed in culture supernatants of Aeromonas hydrophila. The larger protein was isolated by ammonium sulfate precipitation and ion-exchange and hydroxyapatite chromatography and compared with purified aerolysin. It was at least 250 times less hemolytic than aerolysin. Both proteins had the same amino acid sequence at the amino terminus. Cyanogen bromide fragments obtained from the two were identical except that each protein contained one unique fragment, and the fragment from the larger protein was 2,500 daltons larger than the fragment obtained from aerolysin. Treatment with trypsin or with an extracellular Aeromonas protease resulted in rapid conversion of the larger protein to a form corresponding in molecular weight and activity to aerolysin. The results indicate that aerolysin is exported to the culture supernatant as a protoxin which is later activated by proteolytic removal of a peptide from the C terminus.     

The channel-forming toxin aerolysin neutralizes human immunodeficiency virus type 1

Aerolysin is a channel-forming toxin secreted by Aeromonas spp. that binds to glycosyl phosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on sensitive target cells. Receptor binding is followed first by oligomerization of the toxin and then by insertion of the oligomers into the membrane to form stable channels that disrupt the permeability barrier. Human immunodeficiency virus type 1 (HIV-1) produced from T cells is known to incorporate Thy-1 and other GPI-anchored proteins into its membrane. Here, we show that aerolysin is capable of neutralizing HIV-1 in a dose-dependent manner and that neutralization depends upon the presence of these proteins in the viral envelope. Pretreatment with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins greatly reduced HIV-1 sensitivity to the toxin, and virus originating from a mutant cell line that lacks GPI-anchored proteins was not neutralized. Aerolysin variants with single amino acid changes that prevent oligomerization or insertion of the toxin were unable to inactivate the virus, implying that channel formation is necessary for neutralization to occur. These findings represent the first evidence that a pathogenic human virus can be neutralized by a bacterial toxin.