Folding changes in membrane-inserted diphtheria toxin that may play important roles in its translocation.

Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subsequent exposure to the neutral pH of the cytoplasm is believed to aid in translocation of the catalytic A domain of the toxin into the cytoplasm. To understand the effects of low pH and subsequent exposure to neutral pH on translocation, we studied toxin conformation in solution and in toxin inserted in model membranes. Two conformations were found at low pH. One form, L', predominates below 25-30 degrees C, and the other, L", predominates above 25-30 degrees C and is formed from the L' state by an unfolding event. Both forms are hydrophobic and penetrate deeply into membranes. After pH neutralization, the L' and L' conformations give rise to two new conformations, R' and R', respectively. The R' and R" conformations differ from each other in that in the R' state the A domain remains folded, whereas in the R" state the A domain is unfolded. This is confirmed by the finding that only the R' state possesses the capacity to bind and hydrolyze NAD+. It is also supported by the finding that the R' state can also be formed by thermal unfolding of the R' state. The R conformations differ from the low-pH L conformations in that although they remain largely membrane-inserted, it appears that a large portion of the toxin is no longer in contact with the hydrophobic core of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the membrane-inserted diphtheria toxin T domain with peptides and its possible implications for chaperone-like T domain behavior

The T domain of diphtheria toxin is believed to aid the low-pH-triggered translocation of the partly unfolded A chain (C domain) through cell membranes. Recent experiments have suggested the possibility that the T domain aids translocation by acting as a membrane-inserted chaperone [Ren, J., et al. (1999) Science 284, 955-957]. One prediction of this model is that the membrane-inserted T domain should be able to interact with sequences that mimic unfolded proteins. To understand the basis of interaction of the membrane-inserted T domain with unfolded polypeptides, its interaction with water-soluble peptides having different sequences was studied. The membrane-inserted T domain was able to recognize helix-forming 23-residue Ala-rich peptides. In the presence of such peptides, hydrophobic helix 9 of the T domain underwent the previously characterized conformational change from a state exhibiting shallow membrane insertion to one exhibiting deep insertion. This conformational change was more readily induced by the more hydrophobic peptides that were tested. It did not occur at all in the presence a hydrophilic peptide in which alternating Ser and Gly replaced Ala or in the presence of unfolded hydrophilic peptides derived from the A chain of the toxin. Interestingly, a peptide with a complex sequence (RKE(3)KE(2)LMEW(2)KM(2)SETLNF) also interacted with the T domain very strongly. We conclude that the membrane-inserted T domain cannot recognize every unfolded amino acid sequence. However, it does not exhibit strong sequence specificity, instead having the ability to recognize and interact with a variety of amino acid sequences having moderate hydrophobicity. This recognition was not strictly correlated with the strength of peptide binding to the lipid, suggesting that more than just hydrophobicity is involved. Although it does not prove that the T domain functions as a chaperone, T domain recognition of hydrophobic sequences is consistent with it having polypeptide recognition properties that are chaperone-like.     

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.     

Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether

After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1-TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion were studied. These experiments involved bimane or BODIPY labeling of single Cys introduced at various positions, followed by the measurement of bimane emission wavelength, bimane exposure to fluorescence quenchers, and antibody binding to BODIPY groups. Upon exposure of the T domain in solution to low pH, it was found that the hydrophobic face of TH1, which is buried in the native state at neutral pH, became exposed to solution. When the T domain was added externally to lipid vesicles at low pH, the hydrophobic face of TH1 became buried within the lipid bilayer. Helices TH2 and TH3 also inserted into the bilayer after exposure to low pH. However, in contrast to helices TH5-TH9, overall TH1-TH3 insertion was shallow and there was no significant change in TH1-TH3 insertion depth when the T domain switched from the shallowly inserting (P) to deeply inserting (TM) conformation. Binding of streptavidin to biotinylated Cys residues was used to investigate whether solution-exposed residues of membrane-inserted T domain were exposed on the external or internal surface of the bilayer. These experiments showed that when the T domain is externally added to vesicles, the entire TH1-TH3 segment remains on the cis (outer) side of the bilayer. The results of this study suggest that membrane-inserted TH1-TH3 form autonomous segments that neither deeply penetrate the bilayer nor interact tightly with the translocation-promoting structure formed by the hydrophobic TH5-TH9 subdomain. Instead, TH1-TH3 may aid translocation by acting as an A-chain-attached flexible tether.     

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Self-translocation of diphtheria toxin across model membranes.

To understand the mechanism of diphtheria toxin membrane translocation, the toxin was entrapped within lipid vesicles, and its low pH-induced translocation across the lipid bilayer was measured. Proteolysis and resistance to guanidinium chloride denaturation were used to demonstrate that the toxin molecules were entrapped. Low pH-induced movement of entrapped toxin to the outer (trans) face of the bilayer was assayed by the binding of external streptavidin to biotin-labeled entrapped toxin. Complete translocation was quantified by the amount of protein released into the external medium. Using whole toxin, it was found that the A fragment was efficiently translocated, but the B fragment was not. This was true both in the low temperature (A domain folded) and high temperature (A domain unfolded) toxin conformations previously identified [Jiang J. X., Abrams, F. S., and London, E. (1991) Biochemistry 30, 3857-3864]. Remarkably, even isolated fragment A appeared to self-translocate under some conditions. Toxin-induced translocation may partly result from formation of a nonspecific toxin-induced pore. This idea is supported by the toxin-induced release of fluorescent dextrans coentrapped within the vesicles. However, low pH-induced exposure of entrapped toxin on the outside of the membrane was conformation dependent. Exposure was greatest for the high temperature conformation. This suggests the existence of a more specific translocation process. The nature and relationship of these processes, and their relative roles in translocation in vivo are discussed.     

Precursor in cotranslational secretion of diphtheria toxin.

By extracellular labeling of peptides of intact Corynebacterium diphtheriae, followed by fractionation of the cells and chain completion by isolated polysomes, it is shown that diphtheria toxin is formed and secreted cotranslationally by membrane-bound polysomes; free polysomes from none. Moreover, when the chains on these polysomes were completed in vitro, in the absence of membrane they were found to include not only diphtheria toxin of a molecular weight of 62,000, but also a larger precursor of a molecular weight of 68,000. The precursor was identified by several properties: immune precipitation; conversion into toxin fragments A and B; adenosine diphosphate ribosyl-transferase activity after activation with trypsin; and cleavage to 62,000 daltons by membrane enzymes. The precursor yields an N-terminal A fragment with a broadened molecular weight distribution, compared with that from authentic toxin, thus supporting the expectation that the extra segment of the precursor is N-terminal.     

Replacement of negative by positive charges in the presumed membrane-inserted part of diphtheria toxin B fragment. Effect on membrane translocation and on formation of cation channels.

Diphtheria toxin B fragment is capable of forming cation-selective channels in the plasma membrane. Such channels may be involved in the translocation of the toxin A fragment to the cytosol. Seven negatively charged amino acids in the B fragment were replaced one by one by lysines, followed by studies of cytotoxicity and channel-forming ability of the different mutants. The mutant D392K showed a strong reduction in binding to cell surface receptors. Of the six mutants that showed wild-type binding affinity, the two mutants D295K and D318K were very inefficient in forming channels. These two mutants had the lowest ability to mediate A fragment translocation. The mutant E362K was able both to induce cation channel formation and to mediate A fragment translocation at a higher pH value than the wild-type B fragment. The results support the notion that formation of cation channels is of importance for the translocation of the A fragment across the plasma membrane, and they indicate that the pH requirement for translocation of the A fragment to the cytosol is partly determined by the B fragment.     

Action of diphtheria toxin does not depend on the induction of large,stable pores across biological membranes

Summary Vero cells exposed to diphtheria toxin at pH 4.5 leak monovalent cations but not amino acids or phosphorylated metabolites; affected cells do not take up trypan blue. Monovalent cation leakage is inhibited by 1mmCd²⁺, but not by 1mmZn²⁺ or Ca²⁺. Cd²⁺ blocks calcein leakage from liposomes and closes diphtheria toxin-induced channels in lipid bilayers. It is concluded that translocation of the A fragment of diphtheria toxin across biological membranes does not depend on the formation of large stable pores, but that small Cd²⁺-sensitive pores may play a role.     

Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin

When cells with surface-bound diphtheria toxin were exposed to pH 4.5, the toxin became shielded against lactoperoxidase-catalyzed radioiodination, indicating that the toxin was inserted into the membrane. Cells thus treated had strongly reduced ability to take up 36Cl-, 35SO4(2-), and [14C]SCN-. The reduction of chloride uptake was strongest at neutral pH, whereas that of sulfate was strongest at acidic pH. Lineweaver-Burk plots indicated that the toxin treatment reduced the Jmax but not the Km for the anions. The toxin also inhibited the NaCl-stimulated efflux of 35SO4(2-), indicating that the toxin inhibits the antiporter. No inhibition was found when toxin-treated cells were not exposed to low pH, whereas exposure to pH 4.5 for 20 s induced close to maximal inhibition. Half-maximal inhibition was obtained after exposure to pH 5.4. The concentration of diphtheria toxin required to obtain maximal inhibition (0.3 micrograms/ml) was sufficient to ensure close to maximal toxin binding to the cells. Even in ATP-depleted cells and in the absence of permeant anions, low pH induced inhibition of anion antiport in toxin-treated Vero cells. There was no measurable inhibition of anion antiport in cells with little or no ability to bind the toxin.     

Affinity chromatography purification of diphtheria toxin

NAD was covalently linked to Sepharose-4B using a 6 carbon spacer. Sterile, dialyzed spent culture medium containing 100 Lf/ml of diphtheria toxin or material concentrated by (NH₄)₂SO₄ precipitation containing 1500 Lf/ml, was chromatographed on a column of NAD–Sepharose. Ultraviolet absorbing material which did not flocculate with diphtheria antitoxin was eluted with 0.02M phosphate buffer. When the elation buffer was changed to one containing 0.5M NaCl, purified toxin was eluted off the column.     

Structure and function of diphtheria toxin

The survey of the literature on the problem of structural and functional relationship of different parts of diphtheria toxin (i.e. in the binding of toxin to eucaryotic cells receptors, intracellular transport of a-fragment of diphtheria toxin and toxin-mediated ADP-ribosylation of EF2) is presented. Some data concerning structural similarities of A-fragment of diphtheria toxin and C-terminal part of Pseudomonas aeruginosa exotoxin A are presented.     

Characterization of high-order diphtheria toxin oligomers

In 3D domain swapping, a domain of a protein breaks its noncovalent bonds with the protein core and its place is taken by the identical domain of another molecule, creating a strongly bound dimer or higher order oligomer. For some proteins, including diphtheria toxin, 3D domain swapping may affect protein function. To explore the molecular basis of 3D domain swapping in a well-characterized protein system, domain-swapped oligomers of diphtheria toxin were produced by freezing and thawing under a variety conditions, including in various salts and buffers, and at various temperatures. Reaction yields were followed by high-performance size-exclusion chromatography. The traditional low pH pulse produced by freeze-thawing in mixed sodium phosphate buffer induces the oligomerization of DT, but the addition of alkali chloride salts was found to increase the yield in the order of Li(+) > Na(+) > K(+). Unexpectedly, oligomers also formed when DT was frozen and thawed in the presence of 1 M LiCl alone. Slower freezing and thawing of the mixture led to the production of more and larger oligomers. DT oligomers were also produced by exposure to acidic buffers, and were found by electron microscopy to adopt both linear and cyclized forms in a wide distribution of sizes. Upon the basis of these results, the model for the production of DT oligomers by freezing and thawing was expanded to include a salt-mediated pathway. We present a mechanism for the formation of high-order DT oligomers by acidification that takes into account domain swapping and hydrophobic interactions.     

Detoxification of diphtheria and tetanus toxin with formaldehyde. Detection of protein conjugates.

In a model system of purified diphtheria and tetanus toxins it was shown that conjugates between the two proteins are formed during detoxification with formaldehyde. Detoxification mixtures were fractionated by HPLC. Two protein conjugates with different molecular weights were detected and quantified by capture ELISA assay. In vivo the existence of the largest diphtheria-tetanus toxoid conjugate was demonstrated by its antibody response in mice vaccinated with a calcium phosphate adjuvated column fraction of detoxification mixture. To eliminate the risk of cross-linking foreign proteins to toxoids in an attempt to reduce the frequency of adverse reactions in vaccination programmes, it is preferable to purify toxins before treatment with formaldehyde.