Tetanus Neurotoxin Utilizes Two Sequential Membrane Interactions for Channel Formation*

Background: Tetanus neurotoxin enters the neuronal cytosol to block the release of neurotransmitters. Results: Channel formation is enhanced by receptor binding and dependent on acidic lipids. Conclusion: A two-step sequential process takes place: ganglioside-mediated membrane anchorage, followed by pH-triggered channel formation modulated by the membrane environment. Significance: This represents a new paradigm for how A-B toxins translocate enzymatic domains across cellular membranes. Tetanus neurotoxin (TeNT) causes neuroparalytic disease by entering the neuronal soma to block the release of neurotransmitters. However, the mechanism by which TeNT translocates its enzymatic domain (light chain) across endosomal membranes remains unclear. We found that TeNT and a truncated protein devoid of the receptor binding domain (TeNT-LHN) associated with membranes enriched in acidic phospholipids in a pH-dependent manner. Thus, in contrast to diphtheria toxin, the formation of a membrane-competent state of TeNT requires the membrane interface and is modulated by the bilayer composition. Channel formation is further enhanced by tethering of TeNT to the membrane through ganglioside co-receptors prior to acidification. Thus, TeNT channel formation can be resolved into two sequential steps: 1) interaction of the receptor binding domain (heavy chain receptor binding domain) with ganglioside co-receptors orients the translocation domain (heavy chain translocation domain) as the lumen of the endosome is acidified and 2) low pH, in conjunction with acidic lipids within the membrane drives the conformational changes in TeNT necessary for channel formation.

Numerous bacterial pathogens produce toxins that enter into the cytosol of mammalian cells and disrupt normal cellular function. Many of these toxins are referred to as A-B toxins because of their structural and functional organization (1). The B (binding) moiety, composed of one or more subunits, binds to a cell surface receptor and facilitates delivery of the A (active) moiety into the cytosol, where it enzymatically modifies a cellular target. In a subset of A-B toxins, the B domain has the ability to undergo a series of structural changes that allows integration into lipid membranes and formation of a protein conducting channel through which the A domain may be delivered. What drives the structural changes within the B moiety and how polypeptides are translocated across membranes are fundamental questions yet to be fully resolved for any A-B toxin.
The clostridial neurotoxins (CNTs) 2 are a family of bacterial A-B toxins that are among the most lethal natural agents known to humans (2,3). Nine CNTs have been described to date: tetanus neurotoxin (TeNT) produced by Clostridium tetani and eight botulinum neurotoxins (BoNTs, serotypes A-H) produced by strains of Clostridium botulinum, Clostridium butyricum, and Clostridium baratii (4 -7). CNTs are synthesized as single chain polypeptides with a molecular mass of ϳ150 kDa. The precursor is subsequently proteolytically cleaved into an ϳ50-kDa light chain (LC, A subunit) and an ϳ100-kDa heavy chain (HC, B subunit) linked by an essential interchain disulfide bond (8). HC contains an ϳ50-kDa N-terminal translocation domain (HCT) and an ϳ50-kDa C-terminal receptor binding domain (HCR) (9). The HCT facilitates translocation of the LC into the neuronal cytosol, whereas the HCR binds neuronal co-receptors (10 -19).
How CNTs are able to convert from fully folded water-soluble proteins into membrane-integrated protein-translocating channels remains unclear. Traditionally low pH was proposed to trigger the translocation process, presumably by promoting structural changes facilitating the insertion of the HCT into the membrane bilayer. However, the recent demonstration that the isolated HCT of BoNT/A can form ion-conducting channels in the absence of a transmembrane pH gradient brings this model into question (20,21). Rather, it appears that low pH serves to (i) relieve the inhibition of the translocation process mediated by the HCR and (ii) facilitate the partial unfolding of the LC into a conformation necessary for passage through the translocation channel (22,23). The presence of reductant and neutral pH in the cytosol promotes release of the LC from the HC after completion of translocation. Although our understanding of the translocation process has grown in recent years, the precise molecular mechanisms driving the conversion of the watersoluble form into the membrane-integrated form of TeNT remain to be determined.
In the present study, we investigated mechanisms leading to the formation of membrane channels using a combination of full-length TeNT and variants defective in the ability to bind ganglioside co-receptors. Here we demonstrate that ganglioside binding enhances the rate of channel formation, presumably by tethering TeNT close to the target membrane. Furthermore, we demonstrate that membrane association is moderated by the presence of acidic phospholipids, suggesting that the transition from a water-soluble protein into a translocase channel occurs close to the membrane interface. Based on our observations, we propose a sequential two-step model for TeNT channel formation that differs from the mechanisms employed by diphtheria and anthrax toxins, the current paradigms for cell entry of bacterial toxins.
Tetanus Neurotoxin Expression and Purification-DNA encoding TeNT was cloned into the pET28a expression vector (Merck) using appropriate restriction endonuclease sites resulting in an N-terminal His tag fusion protein. To generate a catalytically inactive form of TeNT, two point mutations within the light chain (R372A and Y375F) were generated using the QuikChange II site-directed mutagenesis kit (Agilent). Proteins were expressed in E. coli BL-21 AI cells and purified as described previously (24,25). Peak fractions from the Sephacryl S-200 column were concentrated using an Amicon filtration device (YM-100 type filter), dialyzed into 10 mM HEPES-NaOH, 250 mM NaCl, pH 7.4, and stored at Ϫ80°C until use. A typical preparation yielded 3-5 mg of purified toxin/liter of batch culture.
Cloning and Expression of TeNT LH N Construct-DNA encoding TeNT amino acids 1-864 was amplified by PCR and cloned into the pET-28a expression vector using appropriate restriction endonuclease sites to generate an N-terminal His tag fusion protein. Protein was expressed in E. coli BL-21 AI cells and purified as described for TeNT.
Trypsinization of TeNT Proteins-Trypsin-agarose (500 l) was washed three times in phosphate-buffered saline (PBS) prior to incubation with 2 mg of TeNT or TeNT variants for 60 min at 4°C. Proteins were separated from agarose beads by gentle centrifugation, and a sample was resolved by SDS-PAGE. SDS-PAGE analysis of trypsinized proteins in the presence or absence of reducing agent confirmed that the proteins were converted to dichain molecules of the anticipated sizes (data not shown).

Intoxication of Rat Cortical Neurons with
TeNT-Rat E18 cortical neurons (Brainbits, LLC, Springfield, IL) were cultured on poly-D-lysine-coated glass coverslips in Neurobasal medium supplemented with 0.5 mM Glutamax, Primocin, and B27 supplement for 10 -14 days prior to use. Cells were treated with TeNT or TeNT variants at the indicated concentrations for 24 h at 37°C. In some experiments, cells were pretreated for 1 h with either 100 nM bafilomycin A1 or solvent (DMSO) prior to the addition of the toxins. Following treatment, cells were washed three times in Hanks' balanced salt solution, lysed with radioimmune precipitation assay buffer at 4°C for 30 min, and clarified by centrifugation at 20,000 ϫ g. Lysates were boiled in SDS-PAGE sample buffer, resolved on 13.5% (w/v) SDS-polyacrylamide gels, transferred to Immobilon PVDF membranes, and subjected to Western blotting using antibodies against VAMP2 (1:5000; clone 69.1, Synaptic Systems), ␤-actin (1:1000; mAbcam 8226, Abcam, Cambridge, MA), and goat anti-mouse HRP (1:100,000; Pierce). The membranes were washed, incubated with SuperSignal Dura (Pierce), and visualized using a CCD imaging system. Circular Dichroism-An AVIV model 202 far-UV spectrometer was used to collect spectra (196 -265 nm) of TeNT or TeNT variants (2 M) obtained at pH 7.4 (10 mM HEPES-NaOH, 15 mM NaCl, 0.1 mM EDTA) and pH 4.0 (10 mM sodium acetate-acetic acid, 15 mM NaCl, 0.1 mM EDTA) at 42°C using 1-mm path length quartz cuvettes. Gangliosides were added to a final concentration of 30 M unless otherwise indicated, and samples were rescanned. A spectrum of buffer alone or buffer ϩ gangliosides was subtracted from the appropriate data sets. Data analysis was carried out using CDPro software (26).
Triton X-114 Partitioning-Triton X-114 partitioning assays were performed as described previously (27). For pH 7.4 samples, buffer containing 10 mM HEPES-NaOH, 150 mM NaCl, and 1 mM EDTA was used (hereafter referred to as neutral buffer). For samples at pH 6.5, 6.0, and 5.5, HEPES was replaced with 10 mM MES-NaOH, and for pH 5.0 and 4.0, HEPES was replaced with 10 mM sodium acetate-acetic acid. Final concentrations were 600 nM for TeNT or TeNT variants and 113 M ganglioside. After partitioning, the aqueous and detergent phases of each sample were collected and resolved on 8% (w/v) SDS-polyacrylamide gels, visualized by silver staining, and quantified by densitometry.
Liposome Preparation-Liposomes were freshly prepared by the freeze-thaw and extrusion method as described previously (28). Briefly, lipids (40 mol total) dissolved in chloroform were mixed in the ratios indicated (see the figure legends), dried under a gentle stream of nitrogen, and placed under vacuum overnight to remove residual solvent. The dried lipid cake was hydrated in potassium buffer (10 mM HEPES-KOH, 150 mM KCl, 1 mM EDTA, pH 7.4) to a final concentration of 40 mM by brief sonication at 55°C. The rehydrated lipid was then subjected to three cycles of rapid freeze-thaw, followed by extrusion through a 200-nm pore membrane (Nucleopore) using a miniextruder apparatus (Avanti Polar Lipids). Immediately prior to use, liposomes were exchanged into neutral buffer by passage over a pre-equilibrated column of G-25 Sephadex.
Association and Proteoliposome Isolation-Liposomes (100 l) were combined with 10 g of TeNT or TeNT variants in 500 l of neutral buffer or low pH buffer (10 mM sodium acetateacetic acid, 150 mM NaCl, 1 mM EDTA, pH 4.0). Liposomes were isolated by centrifugation at 100,000 ϫ g for 30 min at 4°C. Supernatants containing unbound proteins were recovered and held on ice for further analysis. Liposomes were suspended in 500 l of fresh neutral or low pH buffer and recovered by centrifugation as above. Supernatants (wash fractions) were collected and, along with those from the first centrifugation step, concentrated to ϳ50 l using Microcon centrifugal filter devices. Liposomes were suspended in 50 l of neutral buffer, and all fractions were mixed with an equal volume of SDS-PAGE buffer. Volume equivalents of each fraction were resolved on SDS-polyacrylamide gels and visualized by staining with Coomassie Blue dye.
K ϩ Release Assay-Liposomes (100 l) were diluted into 5 ml of neutral or low pH buffer with constant stirring and allowed to equilibrate for 1-5 min. TeNT or TeNT variants (2.5 nmol) were then added to the solution, and potassium ion release was monitored using a potassium-selective electrode (Orion, ThermoFisher Scientific). After 5 min, 0.01 M KCl was added to the solution to ensure that the electrode was functioning as expected. Specific K ϩ release was determined by subtraction of basal release values obtained from liposomes incubated in buffer alone.
Statistical Analysis-Densitometric analysis was performed using Protein Simple AlphaView version 3.0 software (Santa Clara, CA). Data were analyzed using GraphPad Prism, version 6.0 (La Jolla, CA). One-way analysis of variance with a Student-Newman-Keuls post-test was performed to determine the difference between means after treatment. Two-way analysis of variance with a Bonferroni post-test was used to determine the difference between pH and treatments and the possible interactions of each. Differences were considered significant at p Ͻ 0.05.

Functional Entry of Recombinant Tetanus Toxin and Protein
Variants into Cortical Neurons-The mechanism by which the LC protease of TeNT is translocated across the endosomal membrane is currently unresolved. To investigate this mechanism further, a series of TeNT variants was constructed (Fig.  1A) and validated by monitoring their ability to enter primary cortical neurons. Exposure of neurons to recombinant TeNT resulted in efficient cleavage of the physiologic substrate VAMP2 after 24 h as determined by Western blotting (Fig. 1B).
To determine whether the receptor binding domain of TeNT is essential for intoxication, a protein variant composed only of the LC and HCT domains was generated (Fig. 1A). When applied to cells, this variant, hereafter named TeNT-LH N , was able to enter cells and cleave VAMP2; however, a significantly higher concentration was needed relative to the full-length toxin. Thus, similar to other A-B type toxins, the receptor bind- ing domain of TeNT is not essential for toxicity. Finally, a recombinant, full-length tetanus toxin was engineered with two point mutations within the light chain (R372A and Y375F). Arg-372 and Tyr-375 are conserved across all CNT family members and are known to contribute to catalysis by facilitating correct alignment of conserved histidine and glutamate residues for the zinc coordination sphere (29 -31). Treatment of cells with a 10,000-fold higher concentration of the catalytic inactive form of toxin (ciTeNT) also failed to cleave VAMP2 (Fig. 1B). This is consistent with the recent study of Blum et al. (32), which showed that mice injected with 5 g of a protein containing the same two mutations (R372A and Y375F, equivalent to ϳ125,000 lethal doses) did not develop any signs of disease. Thus, ciTeNT was substituted for wild type protein in the remaining assays to maximize safety. To further validate the use of TeNT-LH N as a tool to study translocation, the requirement for passage through an acidified compartment was investigated. In agreement with previous studies, the activity of recombinant TeNT was inhibited by the vacuolar proton pump inhibitor bafilomycin A1 (33). TeNT-LH N was also inhibited by bafilomycin A1, indicating that translocation of the light chain by TeNT-LH N was still dependent on exposure to an acidified environment (Fig. 1C).
TeNT Undergoes Secondary Structural Changes in the Presence of Polysialogangliosides at Low pH-Recent studies demonstrated that interaction of ganglioside GT1b with BoNT/B and BoNT/E triggers conformational changes within the two proteins that facilitates transformation into hydrophobic proteins at low pH (34,35). To test whether gangliosides trigger a similar conformational change in TeNT, CD spectroscopy was performed at neutral and acidic pH in the absence and presence of GT1b. At neutral pH, the far-UV CD spectra (196 -265 nm) of TeNT with or without the addition of GT1b overlapped extensively, and the helical content was estimated at 18.4 and 19.6%, respectively ( Fig. 2A and Table 1). Reducing the pH to 4.0 had little effect on the CD spectrum of TeNT, indicating that the protein secondary structure remains largely unchanged. By comparison, the addition of GT1b at low pH caused a dramatic shift in the CD spectrum, with a reduction in helical content to 7.8% and a marked increase in ␤-strand content to 40.8%.
The binding of several CNTs to gangliosides is mediated by a conserved binding pocket located within the HCR (25). It was therefore anticipated that TeNT-LH N , which lacks the HCR, would not undergo secondary structural changes at low pH in the presence of ganglioside. However, similar to full-length TeNT, TeNT-LH N shifted to a largely ␤-strand dominated structure at low pH in the presence of ganglioside ( Fig. 2A and Table 1). It is therefore assumed that the conformational changes occurring in the LC and HCT domains of both proteins are largely the same. Finally, the CD spectra of a mutated TeNT protein deficient in the ability to bind gangliosides (previously termed TeNT RW ) (10, 24) were acquired. The CD spectra of TeNT RW at both neutral and acidic pH are similar to those obtained with the wild-type protein ( Fig. 2 and Table 1). This is consistent with data indicating that mutations at Arg-1226 and Trp-1289 have little effect on the tertiary structure of the HCR (36). Only by the addition of GT1b at low pH could a change in the secondary structure of TeNT RW be observed ( Fig. 2A). Furthermore, pH-triggered conformational changes in TeNT could also be observed by the addition of alternative polysialogangliosides (GD3, GM1a, GD1a, and GD1b) previously demonstrated to bind TeNT in an HCR-dependent manner (data not shown) (24). These data strongly argue against a role for the HCR in detecting the presence of gangliosides at low pH.
Next, the assay was performed using GT1b at concentrations below the reported critical micelle concentration (ϳ1 ϫ 10 Ϫ5 M (37)). Under these conditions, the addition of GT1b did not promote a shift in the CD spectrum of TeNT (Fig. 2B). This implied that structural changes in TeNT might simply be a function of ganglioside micelle formation. If this assertion was correct, then it follows that micelles formed from asialo-GM1a (GA1) should also stimulate a change in TeNT secondary structure. However, as shown in Fig. 2B, the addition of GA1 alone or as mixed micelles composed of GA1 and GT1b (30:1 molar ratio) did not result in secondary structure changes (Fig. 2B). These observations suggest that both micelle formation and the presence of sialic acid(s) are necessary to drive conformational changes in TeNT at low pH.

TeNT Partitions into the Detergent Phase in Triton X-114 Partitioning Assays in the Presence of GT1b at Low pH-Triton
X-114 phase partitioning is routinely exploited as a means of separating hydrophilic proteins from GPI-anchored, acylated, and integral membrane proteins (27). Using this approach, the distribution of TeNT and TeNT-LH N between the aqueous and detergent phases in the presence or absence of ganglioside at pH values ranging from 4.0 to 7.4 was investigated. In the absence of GT1b, TeNT was largely recovered in the aqueous phase. However, the addition of ganglioside caused TeNT to transition from the aqueous to detergent phase only as the pH decreased below ϳ6.5 (Fig. 3A). In comparison, the partitioning of TeNT-LH N into the detergent phase was dependent on the addition of GT1b but unaffected by the pH of the system (Fig.   3B). This observation suggests that the HCR may function in part to mask hydrophobic membrane binding regions until the toxin is exposed to a low pH environment.
TeNT Associates with Liposomal Membranes Enriched in Acidic Lipids-The ability of TeNT to bind to liposomal membranes as a function of pH was determined. Initial experiments employing liposomes composed of zwitterionic lipids (base liposomes, phosphatidylcholine/phosphatidylethanolamine/ cholesterol, 45:45:10, mol %) did not result in detectable binding at either neutral or low pH (data not shown). However, in agreement with previous studies (38,39), pH-dependent binding of both TeNT and TeNT-LH N to asolectin liposomes was observed (Fig. 4A). Based on these opposing observations, it was postulated that the increased anionic charge present in asolectin liposomes might facilitate toxin binding. To directly examine this possibility, base liposomes containing increasing amounts of the acidic phospholipid, phosphatidylserine (PS), were prepared. As shown in Fig. 4B, binding of TeNT to liposomes (base liposomes ϩ PS) at low pH showed a clear depen-  dence on PS through 40 mol %. This supports a role for acidic lipids in triggering membrane association of TeNT. Although binding of TeNT-LH N to liposomes also showed a clear dependence on PS, association with base liposomes (no PS) was also increased relative to TeNT (Fig. 4B). The reason for this difference is not yet clear, but it may reflect exposure of hydrophobic surfaces by removal of the HCR. Acidic Lipids Enhance TeNT-mediated Release of K ϩ from Liposomes-To further characterize the role of acidic lipids in the action of TeNT, channel formation was assayed by measuring K ϩ release from liposomes at neutral and low pH using an ion-specific electrode. The addition of toxins to asolectin liposomes at low pH resulted in a rapid, dose-dependent release of K ϩ content. Treatment of liposomes with identical concentrations of toxins at neutral pH resulted in a small amount of K ϩ release (Ͻ5% of total K ϩ content; data not shown). Consistent with the reported binding data, treatment of base liposomes (zwitterionic) with TeNT or TeNT RW at either neutral or low pH resulted in a minor release of total K ϩ content (Fig. 5, B and  C). A similar level of release was also observed when base liposomes ϩ PS (10 mol %) were exposed to TeNT or TeNT RW at neutral pH (Fig. 5B). Thus, it appears that a basal level of channel formation occurs in a manner largely independent of lipid composition. By comparison, when base liposomes ϩ PS were exposed to TeNT or TeNT RW at low pH, a significant increase in the level of K ϩ release was observed, consistent with the model that membrane binding/channel formation is enhanced by the presence of acidic lipids (Fig. 5C). Given the observed increase in binding of TeNT-LH N to base liposomes (Fig. 4B), it was anticipated that this would also lead to increased K ϩ release at low pH. However, the K ϩ release evoked by TeNT-LH N was similar to that of TeNT and TeNT RW under the tested conditions (Fig. 5, B and C). Thus, at this time we are not able to explain the increased K ϩ release caused by TeNT-LH N from base liposomes containing 10 mol % PS versus base liposomes alone (Fig. 5C).
Binding of TeNT to Liposomes through Ganglioside Receptors Mediates Efficient Channel Formation-To determine what effect cellular receptors may play in the translocation process, base liposomes ϩ PS were doped with gangliosides to facilitate direct binding of TeNT to the membrane. Initial experiments were conducted using liposomes containing 2 mol % GT1b and 10 mol % PS. Under these conditions, the rate of K ϩ release was too rapid to allow for the contribution of GT1b to be assessed (data not shown). Therefore, the concentrations of PS and TeNT were reduced to the minimum levels necessary to detect K ϩ release as compared with base liposomes. Under these conditions (base liposomes ϩ 2 mol % GT1b and 5 mol % PS), binding of TeNT, but not TeNT RW or TeNT-LH N , was observed at both neutral and low pH (Fig. 6A). This demonstrates that binding of TeNT to liposomes containing gangliosides is mediated through HCR-ganglioside interactions and not through electrostatic interactions between the toxin and the charged membrane environment. Coupling of the toxin to the liposomal membrane resulted in significantly enhanced release of K ϩ content when compared with base liposomes containing PS only. However, no such effect was observed when base liposomes ϩ PS and gangliosides were incubated with either TeNT RW or TeNT-LH N (Fig. 6B).

DISCUSSION
CNTs cause neuroparalytic diseases by preventing the release of neurotransmitters at nerve terminals. In recent years, our understanding of their structure-function relationships (e.g. catalytic LC, HCT, and HCR domains), enzymatic mode of action, and identification of neuronal cell surface receptors have all rapidly evolved (9). By comparison, the mechanism by which low pH drives the conversion of CNTs from water-soluble molecules into protein translocase channels remains elusive. Here we investigated the mechanism of channel formation by TeNT to gain new insights into this enigmatic step in the intoxication process. Our data are summarized in Fig. 7, where we propose a novel two-step model for TeNT channel formation.
Initially, the HCR binds to ganglioside co-receptors present in the neuronal plasma membrane (Step 1) (10,24). This interaction presumably promotes partitioning into the bilayer by tethering the HCT close to the membrane, such that its orientation relative to the bilayer is optimal for subsequent channel formation. The interaction of HCR with ganglioside is independent of pH ranging from 4.0 to 7.4 (data not shown), suggesting that the toxin remains bound to the membrane within the acidified endosome. Intriguingly, the interaction of BoNT/B with synaptotagmin II was also reported to be independent of pH, potentially signifying a shared mechanism among the CNTs (40). The need to orient the HCT relative to the membrane may be related to the large ␣-helices of the domain, which probably insert into the bilayer. By comparison, the interaction of diphtheria toxin with its cellular receptors is sensitive to pH, and consequently, formation of membrane channels occurs independently of receptor (41). Furthermore, CNTs do not undergo changes in secondary structure in response to low pH alone (Fig. 2), as has been reported for diphtheria toxin (42). Thus, the initial interaction of TeNT with the membrane does not appear to require the formation of a FIGURE 5. Acidic phospholipids enhance K ؉ release from liposomes. A, asolectin liposomes (4 mol) were diluted into buffer at pH 4.0 and incubated with the indicated proteins (2.5 nmol) for 5 min at 24°C, and K ϩ release was recorded using an ion-specific electrode. K ϩ release values (mV) are the mean Ϯ S.E. (error bars) of three independent experiments. Base liposomes (white bars) or base liposomes ϩ PS (10% PS, mol %; black bars) were diluted into pH 7.4 (B) or pH 4.0 buffer (C) and incubated with the indicated proteins for 5 min at 24°C, and K ϩ release was recorded. K ϩ release values (mV) are the mean Ϯ S.E. of three independent experiments. PC, phosphatidylcholine; PE, phosphatidylethanolamine. FIGURE 6. Ganglioside incorporation enhances K ؉ release from liposomes at low pH. A, ϳ70 pmol of TeNT was mixed with 4 mol of base liposomes ϩ PS and gangliosides (5% PS, 2% mixed gangliosides, mol %) in buffer at either pH 7.4 or pH 4.0. Proteoliposomes were isolated by centrifugation, washed with buffer, and analyzed by SDS-PAGE followed by staining with Coomassie Blue. A representative example of TeNT association with liposomes at pH 7.4 and 4.0 is shown. B, base liposomes ϩ PS (5% PS, mol %; white bars) or base liposomes ϩ PS and gangliosides were diluted into buffer at pH 4.0 and incubated with the indicated proteins (1 nmol) for 5 min at 24°C, and K ϩ release was recorded using an ion-specific electrode. K ϩ release values (mV) are the mean Ϯ S.E. of six independent experiments. Base liposomes ϩ PS and gangliosides were p Ͻ 0.001 (***) or p Ͻ 0.0001 (****) (one-way ANOVA with Student-Newman-Keuls post-test) compared with base liposomes ϩ PS only. PC, phosphatidylcholine; PE, phosphatidylethanolamine; n.s., not significant. membrane-competent state in solution. In step 2, low pH triggers the formation of an interfacial intermediate state that is regulated by the presence of acidic lipids within the membrane. The molecular basis of this regulation is currently unknown and will be the subject of future investigations. The requirement for acidic lipids further distinguishes TeNT from the interaction of diphtheria toxin with the bilayer, which is largely unaltered by the physicochemical nature of the membrane. Once the toxin has formed the interfacial intermediate state, we posit that a rapid and possibly irreversible transformation into the channel state occurs (Fig. 7).
Recent studies have demonstrated that BoNT/B and BoNT/E undergo pH-triggered conformational changes and transformation into oligomeric membrane proteins in the presence of ganglioside GT1b. We observed similar conformational changes in TeNT, a ganglioside-binding deficient form of TeNT (TeNT RW ), and a truncated protein lacking the entire receptor binding domain (TeNT-LH N ) at low pH in the presence of GT1b ( Fig. 2A). These data indicate that the observed structural changes in response to GT1b are not dependent on the HCR. Future studies are planned to determine whether the HCRs of BoNT/B and BoNT/E are necessary for the reported effects of ganglioside on secondary structure and oligomerization. How, therefore, is GT1b able to stimulate the observed changes in TeNT secondary structure? Based on the data presented in Fig. 2, we hypothesize that the observed changes in secondary structure result from insertion of the HCT into the hydrophobic core of the ganglioside micelle. Moreover, given that the addition of GA1 alone or GA1 in combination with a low amount of GT1b did not stimulate a change in secondary structure, the negative charge of the sialic acids also appears to play an important role in the interaction. Thus, it is posited that polysialoganglioside micelles mimic an acidic membrane environment necessary to drive the formation of the interfacial intermediate state as proposed in Fig. 7.
Low pH within the lumen of endosomal compartments has long been postulated as the trigger for translocase channel for-mation. This is in agreement with previous studies demonstrating single-channel activity of BoNTs in planar bilayers and membrane patches excised from neuroendocrine cell lines. Therefore, what role, if any, does lipid composition play in channel formation by BoNTs? Interestingly, channel formation in planar bilayer systems employed either asolectin or defined lipid mixtures containing both phosphatidylserine and ganglioside GT1b (21,43). Thus, the requirement for acidic lipids in regulating the formation of the TeNT interfacial intermediate state may be a shared property among the CNTs. Indeed, Fischer et al. (21) previously noted that translocation activity of BoNT/A devoid of the receptor binding domain could not be observed in non-neuronal cell lines and speculated that membrane lipid composition might contribute to this effect.
In summary, the data presented provide new insights into the mechanism by which the HCT is able to form a channel capable of translocating the LC moiety across the endosomal membrane. Moreover, they suggest a new mechanism for A-B toxin translocation, which differs significantly from the current paradigms of diphtheria and anthrax toxins.