Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.     

Correlation between Shiga toxin B-subunit stability and antigen crosspresentation: a mutational analysis

The homopentameric B-subunit of Shiga toxin (STxB) is used as a tool to deliver antigenic peptides and proteins to the cytosolic compartment of dendritic cells (DCs). In this study, a series of interface mutants of STxB has been constructed. All mutants retained their overall conformation, while a loss in thermal stability was observed. This effect was even more pronounced in trifluoroethanol solutions that mimic the membrane environment. Despite this, all mutants were equally efficient at delivering a model antigenic protein into the MHC class I-restricted antigen presentation pathway of mouse DCs, suggesting that the structural stability of STxB is not a key factor in the membrane translocation process.     

Analysis of Articulation Between Clathrin and Retromer in Retrograde Sorting on Early Endosomes

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans -Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes–TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069–7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.     

Intracellular trafficking of Shiga-toxin-B-subunit-functionalized spherulites

Background information. Spherulites are multi‐lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B‐subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non‐toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures. Results. We demonstrate that STxB‐spherulites (ST×B‐functionalized spherulites) are internalized into HeLa cells in a receptor‐dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane. Conclusions. Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB‐spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB‐spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.     

Direct Pathway from Early/Recycling Endosomes to the Golgi Apparatus Revealed through the Study of Shiga Toxin B-fragment Transport

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37°C, ultrastructural studies on cryosections failed to detect B-fragment–specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor–containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.     

Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum

HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb₃, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, βCOP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed .     

Lipid Reorganization Induced by Shiga Toxin Clustering on Planar Membranes

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb₃ in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb₃ clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb₃ lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb₃ (65∶30∶5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb₃ (40∶35∶20∶5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb₃, STxB-Gb₃ complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.     

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.     

Shiga toxin B-subunit sequential binding to its natural receptor in lipid membranes

Shiga toxin B-subunit (STxB), a protein involved in the cell-binding and intracellular trafficking of Shiga holotoxin, binds to a specific glycolipid, the globotriaosyl ceramide (Gb(3)). Tryptophan residues of STxB, located at the protein-membrane interface, allow one to study its interaction with model membranes by means of spectroscopic methods with no need for chemical derivatisation with a fluorophore. The protein emits maximally around 346 nm and a blue shift of about 8 nm, as well as the occurrence of changes in the emission fluorescence intensity spectra, is indicative of insertion and partition into the membrane. However, the interaction seems to take place without pentamer dissociation. Acrylamide quenching experiments confirm tryptophan residues become less exposed to solvent when in the presence of vesicles, and the use of lipophilic probes suggests that they are located in a shallow position near the water/membrane interface. Fluorescence intensity and lifetime measurements upon STxB titration with Gb(3)-containing vesicles suggest a complex STxB/Gb(3) docking mechanism involving static quenching in the later stages. Based on our observations, a model of the protein-membrane interaction is proposed and the STxB membrane partition and binding constants were calculated.     

Shiga toxin B-subunit sequential binding to its natural receptor in lipid membranes

Shiga toxin B-subunit (STxB), a protein involved in the cell-binding and intracellular trafficking of Shiga holotoxin, binds to a specific glycolipid, the globotriaosyl ceramide (Gb₃). Tryptophan residues of STxB, located at the protein-membrane interface, allow one to study its interaction with model membranes by means of spectroscopic methods with no need for chemical derivatisation with a fluorophore. The protein emits maximally around 346 nm and a blue shift of about 8 nm, as well as the occurrence of changes in the emission fluorescence intensity spectra, is indicative of insertion and partition into the membrane. However, the interaction seems to take place without pentamer dissociation. Acrylamide quenching experiments confirm tryptophan residues become less exposed to solvent when in the presence of vesicles, and the use of lipophilic probes suggests that they are located in a shallow position near the water/membrane interface. Fluorescence intensity and lifetime measurements upon STxB titration with Gb₃-containing vesicles suggest a complex STxB/Gb₃ docking mechanism involving static quenching in the later stages. Based on our observations, a model of the protein-membrane interaction is proposed and the STxB membrane partition and binding constants were calculated.     

Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5 toxin that targets the endoplasmic reticulum chaperone BiP

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB₅ cytotoxins produced by Shiga toxigenic Escherichia coli . Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the endoplasmic reticulum (ER) chaperone BiP. However, its trafficking within target cells has not been investigated previously. In Vero cells, fluorescence colocalization with subcellular markers established that SubAB is trafficked from the cell surface to the ER via a retrograde pathway similar, but not identical, to those of Shiga toxin (Stx) and cholera toxin (Ctx), with their pathways converging at the Golgi. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalization is exclusively clathrin-dependent.     

A cycling cis-Golgi protein mediates endosome-to-Golgi traffic

Toxins can invade cells by using a direct endosome-to-Golgi endocytic pathway that bypasses late endosomes/prelysosomes. This is also a route used by endogenous proteins, including GPP130, which is an integral membrane protein retrieved via the bypass pathway from endosomes to its steady-state location in the cis-Golgi. An RNA interference-based test revealed that GPP130 was required for efficient exit of Shiga toxin B-fragment from endosomes en route to the Golgi apparatus. Furthermore, two proteins whose Golgi targeting depends on endosome-to-Golgi retrieval in the bypass pathway accumulated in early/recycling endosomes in the absence of GPP130. GPP130 activity seemed specific to bypass pathway trafficking because the targeting of other tested proteins, including those retrieved to the Golgi via the more conventional late endosome route, was unaltered. Thus, a distally cycling Golgi protein mediates exit from endosomes and thereby underlies Shiga toxin invasion and retrieval-based targeting of other cycling Golgi proteins.     

The golgin GCC88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-Golgi network

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane-TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.     

Lipid cosorting mediated by shiga toxin induced tubulation

To maintain cell membrane homeostasis, lipids must be dynamically redistributed during the formation of transport intermediates, but the mechanisms driving lipid sorting are not yet fully understood. Lowering sphingolipid concentration can reduce the bending energy of a membrane, and this effect could account for sphingolipid depletion along the retrograde pathway. However, sphingolipids and cholesterol are enriched along the anterograde pathway, implying that other lipid sorting mechanisms, such as protein-mediated sorting, can dominate. To characterize the influence of protein binding on the lipid composition of highly curved membranes, we studied the interactions of the B-subunit of Shiga toxin (STxB) with giant unilamellar vesicles containing its glycosphingolipid receptor [globotriaosylceramide (Gb3)]. STxB binding induced the formation of tubular membrane invaginations, and fluorescence microscopy images of these highly curved membranes were consistent with co-enrichment of Gb3 and sphingolipids. In agreement with theory, sorting was stronger for membrane compositions close to demixing. These results strongly support the hypothesis that proteins can indirectly mediate the sorting of lipids into highly curved transport intermediates via interactions between lipids and the membrane receptor of the protein.     

Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network

An in vitro transport assay, established with a modified Shiga toxin B subunit (STxB) as a marker, has proved to be useful for the study of transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN). Here, we modified this assay to test antibodies to all known soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that have been shown to localize in the Golgi and found that syntaxin 5, GS28, Ykt6, and GS15 antibodies specifically inhibited STxB transport. Because syntaxin 5, GS28, Ykt6, and GS15 exist as a unique SNARE complex, our observation indicates that these four SNAREs function as a complex in EE/RE-TGN transport. The importance of GS15 in EE/RE-TGN transport was further demonstrated by a block in recombinant STxB transport in HeLa cells when GS15 expression was knocked down by its small interfering iRNA. Morphological analyses showed that some GS15 and Ykt6 were redistributed from the Golgi to the endosomes when the recycling endosome was perturbed by SNX3-overexpression, suggesting that GS15 and Ykt6 might cycle between the endosomes and the Golgi apparatus. Further studies indicated that syntaxin 5 and syntaxin 16 exerted their role in EE/RE-TGN transport in an additive manner. The kinetics of inhibition exhibited by syntaxin 16 and syntaxin 5 antibodies is similar.     

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and endoplasmic reticulum (ER). To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes (EE), recycling endosomes, late endosomes and lysosomes. All cargos pass through EE, but may take different routes to the Golgi. Retromer-dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer-dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-mannose-6-phosphate receptor (CI-M6PR), which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CI-M6PR was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the EE, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer-dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB.     

The A-subunit of surface-bound Shiga toxin stimulates clathrin-dependent uptake of the toxin

Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin-receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed.     

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)3 selectively potentiates its photodynamic activity

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)3 into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)3 and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.     

Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit

The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.     

Thermodynamic analysis of the structural stability of the shiga toxin B-subunit

The conformational stability of Shiga toxin B-subunit (STxB), a pentameric protein from Shigella dysenteriae has been characterized by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy under different solvent conditions. It is shown that the thermal folding/unfolding of STxB is a reversible process involving a highly cooperative transition between folded pentamer and unfolded monomers. The conformational stability of STxB is pH dependent and because of its pentameric nature is also concentration dependent. STxB is maximally stable in the pH range from 5 to 9 (Delta G upon unfolding is close to 13 kcal per mol of monomer at 25 degrees C), and its stability decreases both at lower and at higher pH values. The pH dependence of the Gibbs energy of stabilization between pH 2.5 and 5 is consistent with the change in the ionizable state of an average of four groups per monomer upon unfolding. Structural thermodynamic calculations show that the stabilization of the STxB pentamer is primarily due to the interactions established between monomers rather than intramonomer interactions. The folding of an isolated monomer into the conformation existing in the pentamer is unfavorable and expected to be characterized by a free-energy change upon folding in the order of 2.5 kcal mol(-1) at 25 degrees C. On the average, intersubunit interaction induced upon oligomerization of folded monomers should contribute close to -13.4 kcal per mol of monomer to bring the overall Gibbs energy to the experimentally determined value at this temperature.