The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic complex was examined to determine whether it forms an oligomeric prepore complex on the membrane prior to the insertion of its membrane-spanning beta-sheet. A PFO oligomeric complex was formed on liposomes at both 4 degrees C and 37 degrees C and shown by SDS-agarose gel electrophoresis to be comprised of a large, comparatively homogeneous complex instead of a distribution of oligomer sizes. At low temperature, the processes of oligomerization and membrane insertion could be resolved, and PFO was found to form an oligomer without significant membrane insertion of its beta-hairpins. Furthermore, PFO was found to increase the ion conductivity through a planar bilayer by large and discrete stepwise changes in conductance that are consistent with the insertion of a preassembled pore complex into the bilayer. The combined results of these analyses strongly support the hypothesis that PFO forms a large oligomeric prepore complex on the membrane surface prior to the insertion of its transmembrane beta-sheet.     

Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane beta-sheet from a prepore intermediate

Perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin family of pore-forming toxins, forms large oligomeric complexes comprising up to 50 monomers. In the present study, a disulfide bridge was introduced between cysteine-substituted serine 190 of transmembrane hairpin 1 (TMH1) and cysteine-substituted glycine 57 of domain 2 of PFO. The resulting disulfide-trapped mutant (PFO(C190-C57)) was devoid of hemolytic activity and could not insert either of its transmembrane beta-hairpins (TMHs) into the membrane unless the disulfide was reduced. Both the size of the oligomer formed on the membrane and its rate of formation were unaffected by the oxidation state of the Cys(190)-Cys(57) disulfide bond; thus, the disulfide-trapped PFO was assembled into a prepore complex on the membrane. The conversion of this prepore to the pore complex was achieved by reducing the C190-C57 disulfide bond. PFO(C190-C57) that was allowed to form the prepore prior to the reduction of the disulfide exhibited a dramatic increase in the rate of PFO-dependent hemolysis and the membrane insertion of its TMHs when compared with toxin that had the disulfide reduced prior mixing the toxin with membranes. Therefore, the rate-limiting step in pore formation is prepore assembly, not TMH insertion. These data demonstrate that the prepore is a legitimate intermediate during the insertion of the large transmembrane beta-sheet of the PFO oligomer. Finally, the PFO TMHs do not appear to insert independently, but instead their insertion is coupled.     

Monomer-monomer interactions drive the prepore to pore conversion of a beta-barrel-forming cholesterol-dependent cytolysin.

Perfringolysin O (PFO), a cholesterol-dependent cytolysin, forms large oligomeric pore complexes comprised of up to 50 PFO molecules. In the present studies a mutant of PFO (PFO(Y181A)) has been identified that traps PFO in a multimeric prepore complex that cannot insert its transmembrane beta-hairpins and therefore cannot form a pore. Remarkably, PFO(Y181A) can be induced to insert its transmembrane beta-hairpins if functional PFO is incorporated into the PFO(Y181A) oligomeric prepore complex. Furthermore, the transition from prepore to pore appears to be an "all or none" process; partial insertion of the transmembrane beta-barrel does not occur. Therefore, cooperative interactions between the monomers of the prepore drive the prepore to pore conversion that results in the formation of the transmembrane beta-barrel.     

Conformational changes that effect oligomerization and initiate pore formation are triggered throughout perfringolysin O upon binding to cholesterol

Pore formation by the cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target membrane. Cholesterol was long thought to be the cellular receptor for these toxins, but not all CDCs require cholesterol for binding. Intermedilysin, secreted by Streptococcus intermedius, only binds to membranes containing the human protein CD59 but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O (PFO), secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. Given that different steps in the assembly of various CDC pores require cholesterol, here we have analyzed to what extent cholesterol molecules, by themselves, can modulate the conformational changes associated with PFO oligomerization and pore formation. PFO binds to cholesterol when dispersed in aqueous solution, and this binding triggers the distant rearrangement of a beta-strand that exposes an oligomerization interface. Moreover, upon binding to cholesterol, PFO forms a prepore complex, unfolds two amphipathic transmembrane beta-hairpins, and positions their nonpolar surfaces so they associate with the hydrophobic cholesterol surface. The interaction of PFO with cholesterol is therefore sufficient to initiate an irreversible sequence of coupled conformational changes that extend throughout the toxin molecule.     

A New Model for Pore Formation by Cholesterol-Dependent Cytolysins

Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200–300 Å) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three β-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs.     

Prepore to pore transition of a cholesterol-dependent cytolysin visualized by electron microscopy

Perfringolysin O (PFO), a soluble toxin secreted by the pathogenic Clostridium perfringens, forms large homo-oligomeric pore complexes comprising up to 50 PFO molecules in cholesterol-containing membranes. In this study, electron microscopy (EM) and single-particle image analysis were used to reconstruct two-dimensional (2D) projection maps from images of oligomeric PFO prepore and pore complexes formed on cholesterol-rich lipid layers. The projection maps are characterized by an outer and an inner ring of density peaks. The outer rings of the prepore and pore complexes are very similar; however, the protein densities that make up the inner ring of the pore complex are more intense and discretely resolved than they are for the prepore complex. The change in inner-ring protein density is consistent with a mechanism in which the monomers within the prepore complex make a transition from a partially disordered state to a more ordered transmembrane beta-barrel in the pore complex. Finally, the orientation of the monomers within the oligomeric complexes was determined by visualization of streptavidin (SA) molecules bound to biotinylated cysteine-substituted residues predicted to face either the inner or outer surface of the oligomeric pore complex. This study provides an unprecedented view of the conversion of the PFO prepore to pore complex.     

Assembly and topography of the prepore complex in cholesterol-dependent cytolysins

Cholesterol-dependent cytolysins are a family of poreforming proteins that have been shown to be virulence factors for a large number of pathogenic bacteria. The mechanism of pore formation for these toxins involves a complex series of events that are known to include binding, oligomerization, and insertion of a transmembrane beta-barrel. Several features of this mechanism remain poorly understood and controversial. Whereas a prepore mechanism has been proposed for perfringolysin O, a very different mechanism has been proposed for the homologous member of the family, streptolysin O. To distinguish between the two models, a novel approach that directly measures the dimension of transmembranes pores was used. Pore formation itself was examined for both cytolysins by encapsulating fluorescein-labeled peptides and proteins of different sizes into liposomes. When these liposomes were re-suspended in a solution containing anti-fluorescein antibodies, toxin-mediated pore formation was monitored directly by the quenching of fluorescein emission as the encapsulated molecules were released, and the dyes were bound by the antibodies. The analysis of pore formation determined using this approach reveals that only large pores are produced by perfringolysin O and streptolysin O during insertion (and not small pores that grow in size). These results are consistent only with the formation of a prepore complex intermediate prior to insertion of the transmembrane beta-barrel into the bilayer. Fluorescence quenching experiments also revealed that PFO in the prepore complex contacts the membrane via domain 4, and that the individual transmembrane beta-hairpins in domain 3 are not exposed to the nonpolar core of the bilayer at this intermediate stage.     

Specific protein-membrane contacts are required for prepore and pore assembly by a cholesterol-dependent cytolysin

Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. This observation suggested that the D4 loops, which include loops L1-L3 and the undecapeptide, of ILY were no longer required for its cell binding. However, we show here that membrane insertion of the D4 loops is required for the cytolysis by ILY. Receptor binding triggers changes in the structure of ILY that are necessary for oligomerization, but membrane insertion of the D4 loops is critical for oligomer assembly and pore formation. Defects that prevent membrane insertion of the undecapeptide also block assembly of the prepore oligomer, while defects in the membrane insertion of the L1-L3 loops prevent the conversion of the prepore oligomer to the pore complex. These studies reveal that pore formation by ILY, and probably other CDCs, is affected by an intricate and coupled sequence of interactions between domain 4 and the membrane.     

The Cholesterol-dependent Cytolysin Membrane-binding Interface Discriminates Lipid Environments of Cholesterol to Support β-Barrel Pore Insertion

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of β-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the β-barrel pore.     

γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: An AFM Investigation

γ-Hemolysins are bicomponent β-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the stablest one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.     

Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit beta-strand alignment

Cholesterol-dependent cytolysins are bacterial protein toxins that bind to cholesterol-containing membranes, form oligomeric complexes and insert into the bilayer to create large aqueous pores. Membrane-dependent structural rearrangements required to initiate the oligomerization of perfringolysin O monomers have been identified, as have the monomer-monomer interaction surfaces, using site-specific mutagenesis, disulfide trapping and multiple fluorescence techniques. Upon binding to the membrane, a structural element in perfringolysin O moves to expose the edge of a previously hidden beta-strand that forms the monomer-monomer interface and is required for oligomer assembly. The beta-strands that form the interface each contain a single aromatic residue, and these aromatics appear to stack, thereby aligning the transmembrane beta-hairpins of adjacent monomers in the proper register for insertion. Collectively, these data reveal a novel membrane binding-dependent mechanism for regulating cytolysin monomer-monomer association and pore formation.     

The Cholesterol-Dependent Cytolysin Signature Motif: A Critical Element in the Allosteric Pathway that Couples Membrane Binding to Pore Assembly

The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.     

Membrane assembly of the cholesterol-dependent cytolysin pore complex

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced, secreted and contribute to the pathogenesis of many species of Gram-positive bacteria. The assembly of the CDC pore-forming complex has been under intense study for the past 20years. These studies have revealed a molecular mechanism of pore formation that exhibits many novel features. The CDCs form large β-barrel pore complexes that are assembled from 35 to 40 soluble CDC monomers. Pore formation is dependent on the presence of membrane cholesterol, which functions as the receptor for most CDCs. Cholesterol binding initiates significant secondary and tertiary structural changes in the monomers, which lead to the assembly of a large membrane embedded β-barrel pore complex. This review will focus on the molecular mechanism of assembly of the CDC membrane pore complex and how these studies have led to insights into the mechanism of pore formation for other pore-forming proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Mapping the intermedilysin-human CD59 receptor interface reveals a deep correspondence with the binding site on CD59 for complement binding proteins C8alpha and C9

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.     

Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin

Clostridium perfringens epsilon toxin (ETX) rapidly kills MDCK II cells at 37°C, but not 4°C. The current study shows that, in MDCK II cells, ETX binds and forms an oligomeric complex equally well at 37°C and 4°C but only forms a pore at 37°C. However, the complex formed in MDCK cells treated with ETX at 4°C has the potential to form an active pore, since shifting those cells to 37°C results in rapid cytotoxicity. Those results suggested that the block in pore formation at 4°C involves temperature-related trapping of ETX in a prepore intermediate on the MDCK II cell plasma membrane surface. Evidence supporting this hypothesis was obtained when the ETX complex in MDCK II cells was shown to be more susceptible to pronase degradation when formed at 4°C vs. 37°C; this result is consistent with ETX complex formed at 4°C remaining present in an exposed prepore on the membrane surface, while the ETX prepore complex formed at 37°C is unaccessible to pronase because it has inserted into the plasma membrane to form an active pore. In addition, the ETX complex rapidly dissociated from MDCK II cells at 4°C, but not 37°C; this result is consistent with the ETX complex being resistant to dissociation at 37°C because it has inserted into membranes, while the ETX prepore readily dissociates from cells at 4°C because it remains on the membrane surface. These results support the identification of a prepore stage in ETX action and suggest a revised model for ETX cytotoxicity, i) ETX binds to an unidentified receptor, ii) ETX oligomerizes into a prepore on the membrane surface, and iii) the prepore inserts into membranes, in a temperature-sensitive manner, to form an active pore.     

Hydrophobic mismatch is a key factor in protein transport across lipid bilayer membranes via the Tat pathway

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoids, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits: TatA, TatB, and TatC. TatA and TatB possess short transmembrane alpha helices (TMHs), both of which are only 15 residues long in Escherichia coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer, although the functional significance of this mismatch is unclear. Here, we sought to address the functional importance of the hydrophobic mismatch in the Tat transport mechanism in E. coli. We conducted three different assays to evaluate the effect of TMH length mutants on Tat activity and observed that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off following any modification of this length. Surprisingly, TatA and TatB with as few as 11 residues in their TMHs can still insert into the membrane bilayer, albeit with a decline in membrane integrity. These findings support a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, we conclude that the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.     

Structural basis of pore formation by the bacterial toxin pneumolysin

The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands.     

Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin

Perfringolysin O (PFO), a cytolytic toxin secreted by pathogenic Clostridium perfringens, forms large pores in cholesterol-containing membranes. Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition. By using several independent fluorescence techniques, we have determined the topography of D4 in the membrane-inserted oligomeric form of the toxin. Only the short hydrophobic loops at the tip of the D4 beta-sandwich are exposed to the bilayer interior, whereas the remainder of D4 projects from the membrane surface and is surrounded by water, making little or no contact with adjacent protein monomers in the oligomer. Thus, a limited interaction of D4 with the bilayer core seems to be sufficient to accomplish cholesterol recognition and initial binding of PFO to the membrane. Furthermore, D4 serves as the fulcrum around which extensive structural changes occur during the formation and insertion of the large transmembrane beta-barrel into the bilayer.     

Effect of cholesterol and charge on pore formation in bilayer vesicles by a pH-sensitive peptide.

The effect of cholesterol on the bilayer partitioning of the peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) and its assembly into a pore in large unilamellar vesicles composed of neutral and negatively charged phospholipids has been determined. GALA undergoes a conformational change from a random coil to an amphipathic alpha-helix when the pH is reduced from 7.0 to 5.0, inducing at low pH leakage of contents from vesicles. Leakage from neutral or negatively charged vesicles at pH 5.0 was similar and could be adequately explained by the mathematical model (Parente, R. A., S. Nir, and F. C. Szoka, Jr., 1990. Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA. Biochemistry. 29:8720-8728) which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of 10 +/- 2 peptides. Increasing cholesterol content in the membranes resulted in a reduced efficiency of the peptide to induce leakage. Part of the cholesterol effect was due to reduced binding of the peptide to cholesterol-containing membranes. An additional effect of cholesterol was to increase reversibility of surface aggregation of the peptide in the membrane. Results could be explained and predicted with a model that retains the same pore size, i.e., 10 +/- 2 peptides, but includes reversible aggregation of the monomers to form the pore. Resonance energy transfer experiments using fluorescently labeled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol-containing liposomes, thus reducing the efficiency of pore formation.     

Insertion of anthrax protective antigen into liposomal membranes: effects of a receptor

Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K+. Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pH-dependent conformational transition to the pore.