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.     

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.     

Monomer-monomer interactions propagate structural transitions necessary for pore formation by the cholesterol-dependent cytolysins

The assembly of the cholesterol-dependent cytolysin (CDC) oligomeric pore complex requires a complex choreography of secondary and tertiary structural changes in domain 3 (D3) of the CDC monomer structure. A point mutation was identified in the archetype CDC, perfringolysin O, that blocks detectable D3 structural changes and traps the membrane-bound monomers in an early and reversible stage of oligomer assembly. Using this and other mutants we show that specific D3 structural changes are propagated from one membrane-bound monomer to another. Propagation of these structural changes results in the exposure of a β-strand in D3 that allows it to pair and form edge-on interactions with a second β-strand of a free membrane-bound monomer. Pairing of these strands establishes the final geometry of the pore complex and is necessary to drive the formation of the β-barrel pore. These studies provide new insights into how structural information is propagated between membrane-bound monomers of a self-assembling system and the interactions that establish the geometry of the final pore complex.     

Structures of perfringolysin O suggest a pathway for activation of cholesterol-dependent cytolysins

Cholesterol-dependent cytolysins (CDCs), a large family of bacterial toxins, are secreted as water-soluble monomers and yet are capable of generating oligomeric pores in membranes. Previous work has demonstrated that large scale structural rearrangements occur during this transition but the detailed mechanism by which these changes take place remains a puzzle. Despite evidence of structural and functional couplings between domains 3 and 4, the crystal structure of the CDC, perfringolysin O (PFO), shows the two domains do not make direct contact. Here, we present crystal structures of PFO that demonstrate movements of domain 4 are sufficient to trigger conformational changes that are transmitted through the molecule to the distant domain 3. These coupled movements result in a loss of many contacts between domain 3 and rest of the molecule that would eventually lead to the exposure of transmembrane regions in preparation for membrane insertion. The structures reveal a detailed molecular pathway that may be the basis for the allosteric transition that occurs on initial membrane binding leading to the exposure of membrane-spanning regions in a domain distant from the initial site of interaction.     

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.     

Packing a punch: the mechanism of pore formation by cholesterol dependent cytolysins and membrane attack complex/perforin-like proteins

The bacterial cholesterol dependent cytolysins (CDCs) and membrane attack complex/perforin-like proteins (MACPF) represent two major branches of a large, exceptionally diverged superfamily. Most characterized CDC/MACPF proteins form large pores that function in immunity, venoms, and pathogenesis. Extensive structural, biochemical and biophysical studies have started to address some of the questions surrounding how the soluble, monomeric form of these remarkable molecules recognize diverse targets and assemble into oligomeric membrane embedded pores. This review explores mechanistic similarities and differences in how CDCs and MACPF proteins form pores.     

Incomplete pneumolysin oligomers form membrane pores

Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.     

Mouse,but Not Human,ApoB-100 Lipoprotein Cholesterol Is a Potent Innate Inhibitor of Streptococcus pneumoniae Pneumolysin

Streptococcus pneumoniae produces the pore-forming toxin pneumolysin (PLY), which is a member of the cholesterol-dependent cytolysin (CDC) family of toxins. The CDCs recognize and bind the 3β-hydroxyl group of cholesterol at the cell surface, which initiates membrane pore formation. The cholesterol transport lipoproteins, which carry cholesterol in their outer monolayer, are potential off-pathway binding targets for the CDCs and are present at significant levels in the serum and the interstitial spaces of cells. Herein we show that cholesterol carried specifically by the ApoB-100-containing lipoprotein particles (CH-ApoB-100) in the mouse, but not that carried by human or guinea pig particles, is a potent inhibitor of the PLY pore-forming mechanism. Cholesterol present in the outer monolayer of mouse ApoB-100 particles is recognized and bound by PLY, which stimulates premature assembly of the PLY oligomeric complex thereby inactivating PLY. These studies further suggest that the vast difference in the inhibitory capacity of mouse CH-ApoB-100 and that of the human and the guinea pig is due to differences in the presentation of cholesterol in the outer monolayer of their ApoB-100 particles. Therefore mouse CH-ApoB-100 represents a significant innate CDC inhibitor that is absent in humans, which may underestimate the contribution of CDCs to human disease when utilizing mouse models of disease.     

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.     

Characterization of a streptococcal cholesterol-dependent cytolysin with a lewis y and b specific lectin domain

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that often exhibit distinct structural changes that modify their pore-forming activity. A soluble platelet aggregation factor from Streptococcus mitis (Sm-hPAF) was characterized and shown to be a functional CDC with an amino-terminal fucose-binding lectin domain. Sm-hPAF, or lectinolysin (LLY) as renamed herein, is most closely related to CDCs from Streptococcus intermedius (ILY) and Streptococcus pneumoniae (pneumolysin or PLY). The LLY gene was identified in strains of S. mitis, S. pneumoniae, and Streptococcus pseudopneumoniae. LLY induces pore-dependent changes in the light scattering properties of the platelets that mimic those induced by platelet aggregation but does not induce platelet aggregation. LLY monomers form the typical large homooligomeric membrane pore complex observed for the CDCs. The pore-forming activity of LLY on platelets is modulated by the amino-terminal lectin domain, a structure that is not present in other CDCs. Glycan microarray analysis showed the lectin domain is specific for difucosylated glycans within Lewis b (Le (b)) and Lewis y (Le (y)) antigens. The glycan-binding site is occluded in the soluble monomer of LLY but is apparently exposed after cell binding, since it significantly increases LLY pore-forming activity in a glycan-dependent manner. Hence, LLY represents a new class of CDC whose pore-forming mechanism is modulated by a glycan-binding domain.     

Structural Studies of Streptococcus pyogenes Streptolysin O Provide Insights into the Early Steps of Membrane Penetration

Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation–pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane.     

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.     

Intermedilysin-Receptor Interactions during Assembly of the Pore Complex: ASSEMBLY INTERMEDIATES INCREASE HOST CELL SUSCEPTIBILITY TO COMPLEMENT-MEDIATED LYSIS*

Intermedilysin (ILY) is an unusual member of the family of cholesterol-dependent cytolysins because it binds to human CD59 (hCD59) rather than directly to cholesterol-rich membranes. Binding of ILY to hCD59 initiates a series of conformational changes within the toxin that result in the conversion of the soluble monomer into an oligomeric membrane-embedded pore complex. In this study the association of ILY with its membrane receptor has been examined throughout the assembly and formation of the pore complex. Using ILY mutants trapped at various stages of pore assembly, we show ILY remains engaged with hCD59 throughout the assembly of the prepore oligomer, but it disengages from the receptor upon the conversion to the pore complex. We further show that the assembly intermediates increase the sensitivity of the host cell to lysis by its complement membrane attack complex, apparently by blocking the hCD59-binding site for complement proteins C8α and C9.The cholesterol-dependent cytolysins (CDC)² are a family of structurally related pore-forming toxins that are important virulence factors for a variety of Gram-positive pathogens (1–4). The CDCs are secreted by the bacterium as soluble monomers and then bind to cholesterol-rich eukaryotic cell membranes (5). Once bound, the monomers laterally diffuse and interact with one another to form a large oligomeric prepore structure comprised of 35–40 CDC monomers. One of the hallmarks of this family of toxins is the absolute requirement of their pore-forming mechanism on membrane cholesterol (1). Membrane cholesterol serves to target the CDCs to the eukaryotic cell membrane and is necessary to convert the prepore oligomer to the inserted pore complex (6). Two classes of CDCs currently exist. The first class is typified by perfringolysin O (PFO) from Clostridium perfringens that appears to bind directly to cholesterol-rich membranes, an interaction mediated by three short loops in domain 4 (7). The second group includes intermedilysin (ILY) from Streptococcus intermedius and vaginolysin from Gardnerella vaginalis (8). These CDCs bind to the glycosylphosphatidylinositol-anchored protein human CD59 (hCD59). It has been shown for ILY that it first binds hCD59 and then inserts its domain 4 loops in a cholesterol-dependent fashion (7). Why the latter two CDCs have evolved to specifically bind hCD59 and whether they remain engaged with this receptor throughout the assembly of the pore complex remains unclear. S. intermedius is a pathogen frequently associated with abscesses of the oral cavity as well as with life-threatening abscesses of the head, neck, and liver (9, 10). ILY appears to be important in establishing these deep-seated abscesses as S. intermedius isolated from these sites produces levels of ILY 6–10 times greater than strains isolated from peripheral site infections or the oropharynx (9). ILY binds only human cells, whereas other CDCs, such as PFO, bind to most cholesterol-rich eukaryotic membranes. The species selectivity of ILY is because of its specificity for human hCD59 and appears to be encoded in domain 4 of the toxin (11, 12).CD59 is an 18–20-kDa surface-expressed glycoprotein tethered to the cell membrane via a glycosylphosphatidylinositol anchor. It is widely distributed on most human and nonhuman cell types. It is associated with a number of important cellular functions that include serving as an adaptor molecule for a candidate C1q receptor (C1qRO^–₂) (13, 14) and acting as a cell-signaling molecule (15). Its primary role, however, is regulating the terminal pathway of complement by inhibiting the formation of the membrane attack complex (MAC) on host cells by binding to C8α and C9, thus preventing the formation of the MAC pore (16–18). In various autoimmune diseases and inflammatory conditions, excessive complement activation can saturate the available CD59 resulting in MAC-mediated host cell injury (19). CD59 exhibits species selectivity such that it most effectively inhibits only the homologous MAC (20). ILY recognition of the same or similar structural differences in CD59 is the basis for its species selective activity (11).ILY binding to hCD59 triggers a series of conformational changes in ILY leading to its membrane oligomerization into the prepore complex (6). This is accompanied by the cholesterol-dependent insertion of three loops at the base of domain 4 and the insertion of the undecapeptide, events that are necessary for the conversion of the prepore to a pore complex (7). It is not known, however, whether ILY remains engaged with hCD59 throughout its assembly into the pore complex. Whether ILY remains engaged during and after the assembly of the pore complex may also impact the ability of the eukaryotic cell to protect itself from the host MAC because a previous study suggested the ILY-binding site on hCD59 overlaps that for complement proteins C8α and C9 (11). To address these questions, we investigated the interaction of ILY with hCD59 during the assembly of the ILY pore complex. We further determined whether nonlytic assembly intermediates of ILY increase MAC-mediated damage to host cells by short circuiting the protective function of hCD59. These studies show ILY remains engaged during the assembly of its prepore complex and disengages from its receptor upon pore formation. In addition, we show that engagement of hCD59 by ILY prior to pore formation significantly increases the host cell sensitivity to the host MAC-mediated lysis.     

Crystal structure of cytotoxin protein suilysin from Streptococcus suis

Cholesterol-dependent cytolysins (CDC) are pore forming toxins. A prototype of the CDC family members is perfringolysin O (PFO), which directly binds to cholesterol rich cell membrane and lyses the cell. However, as an exception of this general observation, Streptococcus intermedius intermedilysin (ILY) requires human CD59 as its receptor in addition to cholesterol when exhibiting hemolytic activity. It was attempted to explain this functional difference based on a conformational variation in the C-terminal domain of the two toxin proteins, particularly a highly conserved undecapeptide termed tryptophan rich motif. Here, we present the crystal structure of suilysin, a CDC toxin from the swine infectious pathogen Streptococcus suis. Like PFO, suilysin does not require a host receptor for hemolytic activity; yet in the suilysin crystal it shares a similar conformation in the tryptophan rich motif with ILY. This observation suggests that current views of structure-function relationship of CDC proteins in membrane association are still far from complete.     

Cholesterol-dependent cytolysins: from water-soluble state to membrane pore

The cholesterol-dependent cytolysins (CDCs) are a family of bacterial toxins that are important virulence factors for a number of pathogenic Gram-positive bacterial species. CDCs are secreted as soluble, stable monomeric proteins that bind specifically to cholesterol-rich cell membranes, where they assemble into well-defined ring-shaped complexes of around 40 monomers. The complex then undergoes a concerted structural change, driving a large pore through the membrane, potentially lysing the target cell. Understanding the details of this process as the protein transitions from a discrete monomer to a complex, membrane-spanning protein machine is an ongoing challenge. While many of the details have been revealed, there are still questions that remain unanswered. In this review, we present an overview of some of the key features of the structure and function of the CDCs, including the structure of the secreted monomers, the process of interaction with target membranes, and the transition from bound monomers to complete pores. Future directions in CDC research and the potential of CDCs as research tools will also be discussed.     

Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae.

The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.     

Arresting and releasing Staphylococcal alpha-hemolysin at intermediate stages of pore formation by engineered disulfide bonds

alpha-Hemolysin (alphaHL) is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into a heptamer to form a transmembrane pore on a target membrane. The crystal structures of the LukF water-soluble monomer and the membrane-bound alpha-hemolysin heptamer show that large conformational changes occur during assembly. However, the mechanism of assembly and pore formation is still unclear, primarily because of the difficulty in obtaining structural information on assembly intermediates. Our goal is to use disulfide bonds to selectively arrest and release alphaHL from intermediate stages of the assembly process and to use these mutants to test mechanistic hypotheses. To accomplish this, we created four double cysteine mutants, D108C/K154C (alphaHL-A), M113C/K147C (alphaHL-B), H48C/ N121C (alphaHL-C), I5C/G130C (alphaHL-D), in which disulfide bonds may form between the pre-stem domain and the beta-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, alphaHL-A is remarkably stable, is produced at a level at least 10-fold greater than that of the wild-type protein, is monomeric in aqueous solution, and has hemolytic activity that can be regulated by the presence or absence of reducing agents. Cross-linking analysis showed that alphaHL-A assembles on a membrane into an oligomer, which is likely to be a heptamer, in the absence of a reducing agent, suggesting that oxidized alphaHL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of alphaHL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.     

Fiber formation across the bacterial outer membrane by the chaperone/usher pathway

Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform--the usher--is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of gram-negative bacteria.     

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.     

The outer membrane usher forms a twin-pore secretion complex

The PapC usher is an outer membrane protein required for assembly and secretion of P pili in uropathogenic Escherichia coli. P pilus biogenesis occurs by the chaperone/usher pathway, a terminal branch of the general secretory pathway. Periplasmic chaperone-subunit complexes target to the PapC usher for fiber assembly and secretion through the usher to the cell surface. The molecular details of pilus biogenesis at the usher, and protein secretion across the outer membrane in general, are unclear. We studied the structure and oligomeric state of PapC by gel filtration, dynamic light scattering, and electron microscopy and image analysis. Two-dimensional crystals of wild-type PapC and a C-terminal deletion mutant of PapC were produced by reconstituting detergent purified usher into E.coli lipids. PapC formed a dimer both in detergent solution and in the phospholipid bilayer. Cryo-electron microscopy revealed that the usher forms a twin-pore complex. Removal of the C-terminal domain did not change the basic shape of the PapC molecule, but altered the dimeric association of the usher, suggesting that the C terminus forms part of the dimerization interface. The overall molecular size (11 nm), pore size (2 nm), and twin-pore configuration of PapC resemble that of the Tom40 complex, a mitochondrial outer membrane protein translocase.