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Vibrio cholerae cytolysin (VCC) forms SDS-stable heptameric beta-barrel transmembrane pores in mammalian cell membranes. In contrast to structurally related pore formers of gram-positive organisms, no oligomeric prepore stage of assembly has been detected to date. In the present study, disulfide bonds were engineered to tie the pore-forming amino a...
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... IBRIO CHOLERAE cytolysin (VCC) is secreted as a protoxin of 79 kDa (pro-VCC) that requires proteolytic cleavage of an N-terminal fragment to attain pore- forming activity (1, 2). Proteolytic activation can occur both in solution and in the membrane-bound state (3), and only VCC oligomerizes to form SDS-stable heptamers (3, 4, 5). The three-dimensional structure of pro- VCC was solved by Olson und Gouaux (6) and revealed that VCC belongs to the family of  -barrel pore-forming toxins (PFTs; ref. 7). Fluorimetric analyses of the site specifically labeled single cysteine substitution mutant identified the membrane-penetrating amino acid sequence (residues 311–341) as a  -barrel structure (8). The N-terminal proregion exhibits sequence homology with the Hsp90 family and acts as a chaperone (9). The cytolytic domain comprises ϳ 325 amino acids and contains the pore-forming prestem sequence; its structure resembles the water-soluble monomers of Staphylococcus aureus LukF and ␣ -toxin (6, 7, 10). A third  -trefoil domain of ϳ 14 kDa, which shares sequence identity with ricin-like lectin domains, follows the cytolysin domain. The fourth, C-terminal  -prism domain of 15 kDa is absent in related cytolysins from Vibrio vulnificus (10), Aeromonas hydrophila (11), and Aeromonas salmonicida (12). The mechanism of pore assembly and insertion of VCC is poorly characterized. Of particular interest is the fact that assembly of cell-bound VCC into nonlytic oligomeric prepores as described previously for oligomeric PFTs from gram-positive organisms such as ␣ -toxin from S. aureus (13, 14) has not been observed for VCC to date. The process of oligomerization and pore formation requires a series of conformational changes involving protein-protein interactions occur- ring in sequence. To dissect this process, the toxin needs to be trapped at the intermediate stages of pore formation. In this study, disulfide bridges were intro- duced between the prestem and the  -prism or  -trefoil domain, respectively, to prevent rearrangement and membrane insertion of the prestem sequence. Previous work (8) has shown that only 2 of the 6 cysteine residues, C182 and C200, are present in reduced form and that they could be replaced with alanine without loss of activity. Disulfide-bonded mutants constructed from the active C182A/C200A mutant could be ar- rested in a nonlytic prepore state, which could be converted to the functional pore complex by disulfide bond reduction. Mixed oligomers composed of disulfide-trapped mutant and active toxin monomers generated pores with smaller functional diameter, which was interpreted to indicate that only the prestems of the active subunits inserted into the membrane. The col- lective findings are the first documentation that a pore-forming toxin from a gram-negative organism forms oligomeric prepores in the same archetypical manner as originally described for toxins from gram- positive bacteria. All double-cysteine mutants were expressed and purified to homogeneity. The formation of intramolecular disulfide bonds was induced by reacting with diamide and confirmed by SDS-PAGE (Fig. 1 B ). The electrophoretic mobility of the diamide-treated mutants S311C/Q629C*, E322C/Q619C*, and G327C/ A601C* differed slightly from wt* toxin, even if the proteins were heated. Disulfide reduction with DTT normalized the migration behavior in all cases. As shown in Fig. 2 A , the mutants S311C/Q629C*, E322C/Q619C*, and G327C/A601C* were nonhemo- lytic under nonreducing conditions. Strikingly, hemolytic activity of two mutants, E322C/Q619C* and G327C/A601C*, was restored to wt* levels after disulfide reduction (Fig. 2 B ). This indicated that the intro- duced cysteine residues had no effect on the hemolytic activity under reducing conditions and did not disrupt the toxin structure. In contrast to these mutants, S311C/Q629C* showed reduced activity despite the presence of a DTT. For this reason, we focused our research on E322C/Q619C* and G327C/A601C*. The inability of these toxins to lyse erythrocytes in the absence of DTT indicated that the formed disulfide bond prevented assembly into a lytic pore. Lack of hemolytic activity of E322C/Q619C* and G327C/ A601C* could be due to lack of binding or to inability to form the pore; membrane binding was therefore examined as shown in Fig. 3 A . After incubation of either toxin with rabbit erythrocytes at 4°C to prevent oligomerization, unbound toxin was removed by centrifugation, and both the supernatant and the membrane pellet were analyzed by Western blotting. The extent of membrane binding of both G327C/A601C* and E322C/Q619C* mutant toxin was indistinguish- able from wt*. In the experiment conducted with E322C/Q619C*, Coomassie-stained gels of the membrane samples were shown to demonstrate equivalent loading (Fig. 3 A , lanes 6, 8). In the experiment shown in Fig. 3 B , nonreduced and reduced G327C/A601C* or E322C/Q619C* were bound to rabbit erythrocytes at 4°C. Cells incubated with nonreduced toxin were separated from unbound toxin, washed by centrifugation, and then resuspended in PBS or PBS ϩ 5 mM DTT. All samples were incubated at 37°C to allow hemolysis. As shown in Fig. 3 B , the hemolytic activity of either mutant toxin incubated with DTT before binding was the same as for mutant toxin reduced after binding on membranes. The nonreduced mutants again remained hemolytically inactive, although binding was comparable to reduced and fully active toxin (Fig. 3 A ). These results showed that the functional arrest induced by disulfide formation occurred subsequent to membrane binding. To determine whether the disulfide-trapped mutant toxins formed oligomeric prepores, G327C/A601C* was incubated with membranes and analyzed by SDS- PAGE ( Fig. 4 ). Activated toxin was bound to mem- branes at 4°C, separated, and further incubated at 37°C with (lanes 1, 2) or without DTT (lanes 3– 6). The sample without DTT was then divided and incubated without (lanes 3, 4) or with (lanes 5, 6) the cross-linker BS 3 . Addition of DTT to membrane-bound toxin resulted in the formation of SDS-stable oligomers that dissociated at 95°C (lanes 1, 2). However, under nonreducing conditions no band corresponding to the VCC oligomer was observed (lane 3). In contrast, after incubation of cell-bound toxin with the cross-linker BS 3 under nonreducing conditions, oligomers were detected in the absence of DTT (lane 5) and they were no longer dissociated at 95°C (lane 6). Amino acid residue 325 is located in the pore-forming sequence of VCC and directly interacts with the lipid bilayer (8). The integration of the functionally intact acrylodan-labeled G325C* mutant into SDS-stable oligomers could be monitored by UV transillumination of SDS gels and by spectrofluorometry ( Fig. 5 ). A constant amount of acrylodan-labeled G325C* toxin was ad- mixed with increasing amounts of the nonlytic mutant G327C/A601C* and applied to rabbit erythrocyte ghosts. Figure 5 A shows that acrylodan-labeled G325C* toxin alone formed SDS-stabile oligomers on the membranes (lane 1). Increasing amounts of G327C/A601C* added progressively reduced the fraction of SDS-resistant oligomers, indicating that at sufficiently high fractions the disulfide-trapped protein imposed its SDS- sensitive phenotype on the hybrid oligomers (lanes 2–5). Reduction of the mixture shown in lane 5 after binding resulted in formation of SDS-stabile heptamers (lane 6) and revealed that G325C* became incorpo- rated into mixed heptamers. Emission spectra were recorded to examine whether or not the active G325C* subunits located in the hybrid oligomers still retained the ability to insert their prestem domains into the membrane. Membrane insertion of the acrylodan-labeled residue 325 was evident from a blue shift in the fluorescence emission spectrum (8). The extent of the blue shift was virtually indistin- guishable between pure G325C* oligomers and hybrid ones (Fig. 5 B ), even at a 12-fold excess of G327C/ A601C*, which completely suppressed transition of the oligomer to the SDS-resistant state and should statisti- cally result in ...
Citations
... It is needless to say that a precise design of the prestem motif is essential in the context of the -PFT structure and function. Some of the earlier studies have shown that engineered disulphide bond-mediated locking and/or deletion of the pore-forming pre-stem motif block -PFT pore-formation process, and trap the toxins in the pre-pore-like states (24,30,31). Pre-stem motifs of the -PFTs, in general, do not share any J o u r n a l P r e -p r o o f sequence similarity. ...
Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin (β-PFT). Upon binding to the target membranes, VCC monomers first assemble into oligomeric pre-pore intermediates, and subsequently transform into transmembrane β-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane β-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals wild type-like oligomeric β-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature as compared to that of the wild type toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the pre-pore-like intermediate state that is hindered from converting into the functional β-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.
... Water-soluble monomers of VCC bind to the target membranes, and then they assemble into transient pre-pore oligomeric intermediates (Lohner et al., 2009;Paul & Chattopadhyay, 2014;Rai & Chattopadhyay, 2014;Rai & Chattopadhyay, 2015a). Subsequent membrane insertion of the pore-forming motifs creates the functional β-barrel pores (Valeva et al., 2005). ...
β‐barrel pore‐forming toxins perforate cell membranes by forming oligomeric β‐barrel pores. The most crucial step is the membrane‐insertion of the pore‐forming motifs that create the transmembrane β‐barrel scaffold. Molecular mechanism that regulates structural reorganization of these pore‐forming motifs during β‐barrel pore‐formation still remains elusive. Using Vibrio cholerae cytolysin as an archetypical example of the β‐barrel pore‐forming toxin, we show that a key tyrosine residue (Y321) in the hinge region of the pore‐forming motif plays crucial role in this process. Mutation of Y321 abrogates oligomerization of the membrane‐bound toxin protomers, and blocks subsequent steps of pore‐formation. Our study suggests that the presence of Y321 in the hinge region of the pore‐forming motif is crucial for the toxin molecule to sense membrane‐binding, and to trigger essential structural rearrangements required for the subsequent oligomerization and pore‐formation process. Such a regulatory mechanism of pore‐formation by V. cholerae cytolysin has not been documented earlier in the structurally related β‐barrel pore‐forming toxins. Vibrio cholerae cytolysin is a potent membrane‐damaging toxin that perforates cell membranes by forming oligomeric β‐barrel pores. We have shown that a key tyrosine residue in the hinge region of the pore‐forming motif plays a crucial role in regulating the oligomeric pore‐formation mechanism of the toxin. Such a regulatory mechanism has not been documented earlier in the structurally‐related β‐barrel pore‐forming toxins.
... Consistent with generalized mode of action by β-PFT, the pore formation mechanism of HlyA has been proposed to follow three distinct steps ( Figure 2); binding as a water-soluble monomer onto the target cell membrane, formation of pre-pore oligomeric intermediates by the self-assembly of toxin monomer, and finally insertion of the pore-forming stem-loop into the membrane, resulting into the formation transmembrane heptameric β-barrel pores on the cell membrane [78][79][80]. HlyA causes colloid osmotic lysis of mammalian cells by forming transmembrane pores on the target cell membranes [81,82], which causes not only hemolysis but also potent cytotoxic effect such as vacuolation [83] and apoptosis [84,85] of epithelial and immune cells. ...
... However, when osmolytes with hydrodynamic diameters (HDs) above the effective pore size are present, cells are protected by retention of water in the extracellular space. The differential ability of various osmolytes to suppress lysis allows an approximation of pore size, as previously shown for pVCC (37). Osmoprotection assays were carried out with ECPs from the PhlyP-producing strain AR119; pVCC served as a control. ...
Photobacterium damselae subsp. damselae, a pathogenic Vibrio of marine animals may cause septicemia, or hyper-aggressive necrotizing fasciitis in humans. Previously we have shown that hemolysin genes are critical for virulence of this organism in mice and fish. In co-culture experiments we found that photobacteria led to rapid permeabilization of epithelial cells for vital dyes. This effect could be largely attributed to the product of plasmid encoded hlyA, a putative small β-pore forming toxin. Here we have characterized this new hemolysin and termed it phobalysin P (PhlyP) for photobacterial lysin encoded on a plasmid. PhlyP formed stable oligomers and small membrane pores. It caused massive efflux of K+, no significant leakage of lactate dehydrogenase but entry of vital dyes; the latter distinguishes PhlyP from related Vibrio cholerae cytolysin. Attack by PhlyP provoked rapid loss of cellular ATP, attenuated translation, and provoked profound morphological changes in epithelial cells. Unexpectedly, photobacterial hemolysins promoted bacterial association with target cells. Similar observations with other hemolysins, target cells and bacteria suggest that this is a common effect of membrane damaging toxins.
http://iai.asm.org/content/early/2015/08/20/IAI.00277-15.abstract
... In water-soluble monomers, these loops are folded up against the body of the monomer to prevent the energetically costly exposure of hydrophobic surfaces to aqueous solution. How water-soluble monomers assemble to form the final channel is less well understood, but the generally accepted paradigm based on studies of staphylococcal ␣-hemolysin (14) and applicable to VCC (15) suggests that watersoluble monomers bind individually to membranes via interactions with the membrane-contacting rim domain (Fig. 1A), diffuse and assemble into oligomeric non-lytic "prepores," and then cooperatively insert their amphipathic loops into the membrane to form the final -barrel. It is important to note that VCC and structurally related toxins are only one subset of a much larger group of structurally dissimilar classes of PFTs, which have been extensively reviewed elsewhere (16). ...
Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell-surface receptors that increase the local toxin concentration on cell membranes thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. While it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell-lines we show that VCC is unlikely to utilize high-affinity protein receptors like structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino-acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan-binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding-interactions.
... hemolysin | streptolysin | sterol | toxin | alpha-hemolysin T he cholesterol-dependent cytolysins (CDCs) make up the largest class of bacterial β-barrel pore-forming toxins (βPFTs) and are present in nearly 50 Gram-positive opportunistic pathogens. A central paradigm of βPFTs is the formation of an obligatory intermediate termed the prepore (1)(2)(3)(4)(5)(6). The prepore is a membrane-bound oligomerized ring-shaped complex in which the membrane-spanning β-barrel pore has not formed. ...
Significance
Bacterial pathogens produce pore-forming toxins that damage eukaryotic membranes, whereas the pore-forming immune defense proteins produced by vertebrates can damage bacterial membranes. Despite the opposite functions of these proteins in pathogenesis or protection, many use a common pore-forming mechanism whereby membrane-bound monomers oligomerize into a circular structure, termed the prepore, which then assembles a β-barrel structure that punches a hole in the membrane. Here we show that once the prepore is assembled, an intermolecular electrostatic interaction is established that drives the formation of the pore. This mechanism is likely to be used by toxins and other pore-forming proteins that span the biological domains of life.
... The crystal structures of g-HL-WR and LUK avoided such steric hindrance, as the stem regions were not folded but extruded to form the upper half of the b-barrel (Figs 1a and 4a). Prepore state oligomers have been trapped previously using mutants in which disulphide linkage was artificially introduced between prestem and cap domain or within prestem 12,28,29 . Most of these oligomers were weakly assembled, and were observed only in the presence of chemical crosslinker 29 . ...
Pathogenic bacteria secrete pore-forming toxins (PFTs) to attack target cells. PFTs are expressed as water-soluble monomeric proteins, which oligomerize into nonlytic prepore intermediates on the target cell membrane before forming membrane-spanning pores. Despite a wealth of biochemical data, the lack of high-resolution prepore structural information has hampered understanding of the β-barrel formation process. Here, we report crystal structures of staphylococcal γ-haemolysin and leucocidin prepores. The structures reveal a disordered bottom half of the β-barrel corresponding to the transmembrane region, and a rigid upper extramembrane half. Spectroscopic analysis of fluorescently labelled mutants confirmed that the prepore is distinct from the pore within the transmembrane region. Mutational analysis also indicates a pivotal role for the glycine residue located at the lipid-solvent interface as a 'joint' between the two halves of the β-barrel. These observations suggest a two-step transmembrane β-barrel pore formation mechanism in which the upper extramembrane and bottom transmembrane regions are formed independently.
... VCC is a pore-forming toxin that induces lysis of different mammalian cells by forming transmembrane 14-stranded b-barrel channels in the plasma membrane [50]. At sub-lytic concentration, VCC can trigger vacuolization and apoptosis of epithelial and immune cells [14,51]. ...
Background:
Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.
Methodology/principal findings:
OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.
Conclusion/significance:
Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.
... Consistent with the generalized -PFT mode of action, the mechanism of membrane pore formation by VCC is proposed to follow three distinct steps: binding of the toxin monomers onto the target cell membrane; formation of transient, metastable prepore oligomeric intermediates on the membrane; and conversion of the prepore oligomers into the transmembrane oligomeric  barrel pores (6, 8, 14 -17). Studies on several -PFTs, including VCC, also suggest that the formation of the functional transmembrane oligomeric pore structure involves membrane insertion of the pore-forming stem loop from each of the toxin protomers toward generation of the transmembrane  barrel segments (18,19). However, it has not been tested experimentally, at least in the case of VCC, whether the membrane insertion of the stem loop could occur in the membrane-bound monomeric state before the prepore oligomer formation or whether the prepore oligomer formation precedes membrane insertion. ...
... Even in the case of generalized -PFT mechanisms, such a sequence of events has not been established unambiguously. Previous studies have employed engineered -PFT variants (for example, staphylococcal LukF and VCC) incapable of inserting their pore-forming stem loop into the membrane lipid bilayer (18,20). Such toxin variants, having their stem loop in a locked configuration via engineered disul-fide linkage, are found to remain trapped in their prepore oligomeric state (18,20). ...
... Previous studies have employed engineered -PFT variants (for example, staphylococcal LukF and VCC) incapable of inserting their pore-forming stem loop into the membrane lipid bilayer (18,20). Such toxin variants, having their stem loop in a locked configuration via engineered disul-fide linkage, are found to remain trapped in their prepore oligomeric state (18,20). These observations, however, do not address the issue whether oligomerization is absolutely essential to initiate membrane insertion or whether membrane insertion could be initiated before prepore formation. ...
Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytolytic toxin that belongs to the family of β-barrel pore-forming protein toxins (β-PFTs). VCC induces lysis of its target eukaryotic cells by forming transmembrane oligomeric β-barrel pores. Mechanism of membrane pore formation by VCC follows overall scheme of the archetypical β-PFT mode of action, in which water-soluble monomeric form of the toxin first binds to the target cell membrane, then assembles into a pre-pore oligomeric intermediate, and finally converts into the functional transmembrane oligomeric β-barrel pore. However, there exists a vast lacuna in our understanding regarding the intricate details of the membrane pore-formation process employed by VCC. In particular, membrane oligomerization and membrane insertion steps of the process have been described only to a limited extent. In the present study, we have determined the key residue(s) in VCC that are critical to trigger membrane oligomerization of the toxin. Alteration of such key residue(s) traps the toxin in its membrane-bound monomeric state, and abrogates subsequent oligomerization, membrane insertion, and functional transmembrane pore-formation events. Results obtained from our study also suggest that the membrane insertion of VCC depends critically on the oligomerization process, and it cannot be initiated in the membrane-bound monomeric form of the toxin. In sum, our study for the first time dissects membrane binding from the subsequent oligomerization and membrane insertion steps, and thus defines the exact sequence of events in the course of membrane pore formation process by VCC.
... Vibrio cholerae cytolysin/hemolysin (VCC) 3 is a water-soluble -pore-forming toxin (-PFT) (1-3) with a native molecular mass of 65 kDa (4).VCC, which induces lysis of mammalian cells by forming transmembrane 14-stranded -barrel channels 0.9 nm in diameter in the plasma membrane bilayer (3,5), triggers vacuolation (6) and apoptosis (7,8) of epithelial and immune cells at sublytic concentrations. VCC, which is enterotoxic in rabbit ligated ileal loop (9) and lethal in mouse pulmonary models (10), is widely thought to enhance virulence of V. cholerae, the organism responsible for human cholera (11). ...
... Vibrio cholerae cytolysin/hemolysin (VCC) 3 is a water-soluble -pore-forming toxin (-PFT) (1-3) with a native molecular mass of 65 kDa (4).VCC, which induces lysis of mammalian cells by forming transmembrane 14-stranded -barrel channels 0.9 nm in diameter in the plasma membrane bilayer (3,5), triggers vacuolation (6) and apoptosis (7,8) of epithelial and immune cells at sublytic concentrations. VCC, which is enterotoxic in rabbit ligated ileal loop (9) and lethal in mouse pulmonary models (10), is widely thought to enhance virulence of V. cholerae, the organism responsible for human cholera (11). ...
Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65 kDa β-pore-forming toxin with a carboxyl-terminus β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromised hemolytic activity, it is thought that carbohydrate-dependent interactions played a critical role in membrane-targeting of VCC. To delineate contributions of the cytolysin and lectin domains in pore formation, we used the wild-type VCC, the 50 kDa VCC(50) without the lectin domain and the mutant VCC(Asp617Ala) with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants of the order of 10(7) M(-1). However, loss of the lectin domain severely reduced efficiency of self-association of the VCC monomer to the β-barrel heptamer in synthetic lipid bilayer from ~83 to 27%. Notably, inactivation of the carbohydrate-binding activity by Asp(617)Ala mutation marginally reduced oligomerization to ~77%. Oligomerization of VCC(50) was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process was driven by entropy and opposed by enthalpy. Asialofetuin, the β1-galactosyl-terminated glycoprotein inhibitor of VCC induced hemolysis, promoted oligomerization of the 65 kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose: (i) the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer, and (ii) glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.
