Concerted protonation of key histidines triggers membrane interaction of the diphtheria toxin T domain

The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His(223)-His(257) and His(251) are the most sensitive triggers for the formation of the molten globule state in solution, whereas His(322)-His(323) and His(251) are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.     

Behavior of the deeply inserted helices in diphtheria toxin T domain: helices 5, 8, and 9 interact strongly and promote pore formation, while helices 6/7 limit pore formation

Diphtheria toxin T domain aids the membrane translocation of diphtheria toxin A chain. When the isolated T domain is deeply membrane-inserted, helices TH 8-9 form a transmembrane hairpin, while helices TH 5-7 form a deeply inserted nontransmembrane structure. Blocking deep insertion of TH 8-9 blocks deep insertion of TH 5-7 ( Zhao, G., and London, E. ( 2005) Biochemistry 44, 4488- 4498 ). We now examine the effects of blocking the deep insertion of TH 5 and TH 6/7. An A282R/V283R dual substitution in TH 5 prevented its deep insertion, significantly decreased the deep insertion of TH 9, and to a lesser degree that of TH 6/7. Blocking deep insertion of TH 6/7 with a L307R mutation had no effect on the deep insertion of TH 5, similar to its previously characterized lack of effect on the deep insertion of TH 8-9. An I364K mutation in TH 9 blocked TH 8-9 deep insertion and greatly reduced pore formation by the T domain, consistent with the role of TH 8-9 in pore formation. The A282R/V283R mutations also reduced the extent of pore formation, but to a lesser degree, suggesting either that TH 5 is part of the pore or that interactions with TH 5 affect the ability of TH 8-9 to form pores. The L307R mutation enhanced the extent of pore formation, suggesting that deeply inserted TH 6/7 may act as a "cork" that partly blocks the pore. Combined, these results indicate that TH 5, 8, and 9 combine to form a deeply inserted scaffold of more strongly associated helices.     

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.     

Accessibility changes within diphtheria toxin T domain upon membrane penetration probed by hydrogen exchange and mass spectrometry

The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the pH inside endosomes is acid. At that stage, the domain is in a partially folded state; this prevents the use of high-resolution methods for the characterization of its functional structure. On that purpose, we report here the use of hydrogen/deuterium exchange experiments coupled to mass spectrometry. The conformation changes during the different steps of insertion into lipid bilayer are monitored with a resolution of few residues. Three parts of the translocation domain can be distinguished. With a high protection against exchange, the C-terminal hydrophobic helical hairpin is embedded in the membrane. Despite a lower protection, a significant effect in the presence of lipid vesicles shows that the N-terminal part is in interaction with the membrane interface. The sensitivity to the ionic strength indicates that electrostatic interactions are important for the binding. The middle part of the domain has an intermediate protection; this suggests that this part of the domain can be embedded within the membrane but remains quite dynamic. These results provide unprecedented insight into the structure reorganization of the protein to go from a soluble state to a membrane-inserted one.     

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.     

The number of subunits comprising the channel formed by the T domain of diphtheria toxin.

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine(+) and NAD(-), it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an NH(2)-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric.     

Creation of intercellular bonds by anchoring protein ligands to membranes using the diphtheria toxin T domain

We describe the creation of cell adhesion mediated by cell surface engineering. The Flt3-ligand was fused to a membrane anchor made of the diphtheria toxin translocation domain. The fusion protein was attached to the surface of a cell by an acid pulse. Contact with another cell expressing the receptor Flt3 lead to its activation. This activity involved direct cell-cell contact. A mean force of 20 nN was needed to separate functionalized cells after 5 min of contact. Overall, we showed that it is possible to promote specific cell-cell adhesion by attaching protein ligands at the surface of cells.     

Extension of juxtamembrane domain of diphtheria toxin receptor arrests translocation of diphtheria toxin fragment A into cytosol

Diphtheria toxin (DT) binds to the EGF-like domain of the DT receptor (DTR), followed by internalization and translocation of the enzymatically active fragment A into the cytosol. The juxtamembrane domain (JM) of the DTR is the linker domain connecting the transmembrane and EGF-like domains. We constructed mutants of DTRs with altered JMs and studied their abilities for DT intoxication. Although DTR mutants with extended JMs showed normal DT binding activity, the cells expressing the mutants showed both reduced translocation of DT fragment A into the cytosol and reduced sensitivity to DT, when compared with cells expressing wild-type DTR. These results indicate that the JM contributes to DT intoxication by providing a space appropriate for the interaction of DT with the cell membrane. The present study also indicates that consideration of epitopes of an immunotoxins would be an important factor in the design of potent immunotoxins.     

Ion channel and membrane translocation of diphtheria toxin

Abstract Diphtheria toxin is the best studied member of a family of bacterial protein toxins which act inside cells. To reach their cytoplasmic targets, these toxins, which include tetanus and botulinum neurotoxins and anthrax toxin, have to cross the hydrophobic membrane barrier. All of them have been shown to form ion channels across planar lipid bilayer and, in the case of diphtheria toxin, also in the plasma membrane of cells. A relation between the ion channel and the process of membrane translocation has been suggested and two different models have been put forward to account for these phenomena. The two models are discussed on the basis of the available experimental evidence and in terms of the focal points of difference, amenable to further experimental investigations.     

Behavior of diphtheria toxin T domain containing substitutions that block normal membrane insertion at Pro345 and Leu307: control of deep membrane insertion and coupling between deep insertion of hydrophobic subdomains

Diphtheria toxin T domain aids the translocation of toxin A chain across membranes. T domain has two hydrophobic layers/subdomains that can insert deeply into membranes: helices TH8 and 9, which form a transmembrane hairpin, and helices TH5-7, which form a nonclassical, nontransmembrane structure. Substitutions were made at Pro345, a residue located near the turn between TH8 and 9. P345 is critical for toxicity and pore formation by the T domain. Fluorescence methods showed that hairpin-disrupting Gly or Glu substitutions at 345 did not insert into lipid bilayers as deeply as the wild-type protein, and consistent with previous studies, these mutations reduced pore formation activity as assayed by a novel biotin-streptavidin-based influx assay. Introducing Pro at positions 347 or 353 not only failed to compensate for substitutions at P345, but also they further disrupted deep insertion and/or pore formation. Substitution of P345 with Asn, a residue that promotes helical hairpin formation almost as well as Pro, resulted in somewhat more normal insertion and pore formation than other substitutions. Importantly, a P345E substitution disrupted deep insertion of TH5-7. This suggests that TH8 and 9 and TH5-7 undergo some sort of coordinated insertion into the lipid bilayer and/or that the membrane-inserted T domain has a distinct tertiary structure in which TH5-7 interact with TH8 and 9 instead of consisting of noninteracting hydrophobic segments. Intriguingly, a L307R substitution in TH6, which disrupted deep insertion of TH7, had only a weak effect on pore formation and deep insertion of TH8 and 9. This suggests that the TH8 and 9 region can insert independently of TH5-7 to some degree and that TH8 and 9 insertion may occur early in T-domain insertion.     

Single mutation in the A domain of diphtheria toxin results in a protein with altered membrane insertion behavior

The insertion of the A domain of diphtheria toxin into model membranes has been shown to be both pH- and temperature-dependent (Hu and Holmes (1984) J. Biol. Chem. 259, 12226-12233). In this report, the insertion behavior of two mutant proteins of diphtheria toxin, CRM197 and CRM9, was studied and compared to that of wild-type toxin. Results indicated that both CRM197 and CRM9 resembled toxin with respect to the pH-dependence of binding to negatively-charged liposomes at room temperature. However, CRM197 differed from toxin with respect to both the pH- and temperature-dependence of fragment A insertion; fragment A197 inserts more readily into the bilayer at 0 degrees C and low pH or at neutral pH and room temperature than does wild type fragment A under these same conditions. This result indicates that the single amino acid substitution in the A domain of CRM197 facilitates entry of fragment A197 into the membrane, suggesting that CRM197 may be conformationally distinct from native toxin. In fact, the fluorescence spectra of CRM197 and wild-type toxin as well as their respective tryptic peptide patterns indicate that, at pH 7, CRM197 more closely resembles the acid form of wild-type toxin than the native form of toxin. These data suggest that CRM197 may be naturally in a more 'insertion-competent' conformation. In contrast, the mutation in the B domain of CRM9 which results in a 1000-fold decrease in binding affinity for plasma membrane receptors apparently does not cause a change in either the insertion of fragment A9 or the lipid-binding properties of CRM9 relative to toxin.     

Topography of helices 5-7 in membrane-inserted diphtheria toxin T domain: identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin

The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes. T domain hydrophobic helices 8 and 9 can adopt two conformations, one close to the membrane surface (P state) and a second in which they apparently form a transmembrane hairpin (TM state). We have now studied T domain helices 5-7, a second cluster of hydrophobic helices, using Cys-scanning mutagenesis. After fluorescently labeling a series of Cys residues, penetration into a non-polar environment, accessibility to externally added antibodies, and relative depth in the bilayer were monitored. It was found that helices 5-7 insert shallowly in the P state and deeply in the TM state. Thus, the conformational changes in helices 5-7 are both similar and somehow linked to those in helices 8 and 9. The boundaries of deeply inserting sequences were also identified. One deeply inserted segment was found to span residues 270 to 290, which overlaps helix 5, and a second spanned residues 300 to 320, which includes most of helix 6 and all of helix 7. This indicates that helices 6 and 7 form a continuous hydrophobic segment despite their separation by a Pro-containing kink. Additionally, it is found that in the TM state some residues in the hydrophilic loop between helices 5 and 6 become more highly exposed than they are in the P state. Their exposure to external solution in the TM state indicates that helices 5-7 do not form a stable transmembrane hairpin. However, helix 5 and/or helices 6 plus 7 could form transmembrane structures that are in equilibrium with non-transmembrane states, or be kinetically prevented from forming a transmembrane structure. How helices 5-7 might influence the mechanism by which the T domain aids translocation of the diphtheria toxin A chain across membranes is discussed.     

The interaction between actin and FA fragment of diphtheria toxin

Actin protein has many other cellular functions such as movement, chemotaxis, secretion and cytodiaresis. Besides, it have structural function. Actin is a motor protein that it has an important role in the movement process of toxin in the cell. It is known that F-actin gives carriage support during the endosomal process. Actin is found in globular (G) and filamentous (F) structure in the cell. The helix of actin occurs as a result of polymerisation of monomeric G-actin molecules through sequential rowing, is called F-actin (FA). Actin interacts with a great number of cellular proteins along with cell skeleton and plasma membrane. It is also known that some bacterial toxins have ADP-ribosylation affect on actin. Diphteria toxin is the part which has the FA enzymatic activity corresponding the N-terminal section of the toxin, which inhibits the protein synthesis by ADP-ribosylating the elongation factor 2 in the presence of NAD. FA, taken into the cell by endocytosis inhibits protein synthesis by ADP-ribosyltransferase activity and breaks the cytoskeleton. In the studies both in vitro and in vivo, actin with interaction FA of diphteria toxin has been yet to be fully elucidated. The aim of this study was to determine the three dimensional structures of actin with interaction FA of diphteria toxin by the amprical methods and in paralel with the computing technology, theoretical methods have gained significant importance. In our study, actin with interaction FA of diphteria toxin has been determined as the most possible interaction area with the theoretical method; analogy modelling. This area has been closed in the presence of polypeptides and FA-actin interactions have been tested with the gel filtration chromatography techniques. As a result of the findings, we found that 15 amino acid artificial peptides (DAMYETMAQACAGNR) corresponding to 201–215 amino acid residues of FA interacts with G-actin and closes this area. Secondly, in the model formed with the analogy modelling, it appears that the most possible interaction area is between FA (tyr204) and G-actin (gly48). Results obtained from both theoretical and experimental data support the idea that the interaction occurs in this area.     

Localization of the diphtheria toxin receptor-binding domain to the carboxyl-terminal Mr approximately 6000 region of the toxin

The carboxyl-terminal Mr = 5982 peptide of diphtheria toxin was prepared by specific cleavage of the toxin with hydroxylamine and purified by fast performance liquid chromatography. The identity of the peptide was established by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, reactivity with specific monoclonal antibodies, and amino-terminal sequence analysis. The Mr = 5982 peptide was shown to protect highly toxin-sensitive Vero cells from the lethal action of diphtheria toxin. This protection was shown to be due to inhibition of the initial step in the cytotoxic process, the binding of toxin to its receptor. These results strongly suggest that the Mr = 5982 carboxyl-terminal region (amino acid residues 482-535) is, or contains, the receptor-binding domain of diphtheria toxin.     

Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-streptavidin: at low pH, helices 8 and 9 form a transmembrane hairpin but helices 5-7 form stable nonclassical inserted segments on the cis side of the bilayer

Low pH-induced membrane insertion by diphtheria toxin T domain is crucial for A chain translocation into the cytoplasm. To define the membrane topography of the T domain, the exposure of biotinylated Cys residues to the cis and trans bilayer surfaces was examined using model membrane vesicles containing a deeply inserted T domain. To do this, the reactivity of biotin with external and vesicle-entrapped BODIPY-labeled streptavidin was measured. The T domain was found to insert with roughly 70-80% of the molecules in the physiologically relevant orientation. In this orientation, residue 349, located in the loop between hydrophobic helices 8 and 9, was exposed to the trans side of the bilayer, while other solution-exposed residues along the hydrophobic helices 5-9 region of the T domain located near the cis surface. A protocol developed to detect the movement of residues back and forth across the membranes demonstrated that T domain sequences did not rapidly equilibrate between the cis and the trans sides of the bilayer. Binding streptavidin to biotinylated residues prior to membrane insertion only inhibited T domain pore formation for residues in the loop between helices 8 and 9. Pore formation experiments used an approach avoiding interference from transient membrane defects/leakage that may occur upon the initial insertion of protein. Combined, these results indicate that at low pH hydrophobic helices 8 and 9 form a transmembrane hairpin, while hydrophobic helices 5-7 form a nonclassical deeply inserted nontransmembraneous state. We propose that this represents a novel pre-translocation state that is distinct from a previously defined post-translocation state.     

Conformation and model membrane interactions of diphtheria toxin fragment A

Low pH is believed to play a critical role in the penetration of membranes by diphtheria toxin in vivo. In this report, the pH dependence of the conformation of fragment A of diphtheria toxin has been studied using fluorescence techniques. As pH is decreased, fragment A in solution undergoes a reversible conformational change beginning below pH 5. The conformational change occurs rapidly upon exposure to low pH. It involves both an increase in the exposure of tryptophanyl residues to solution and a switch from a hydrophilic state to a hydrophobic state as judged by fragment A binding to micelles of a mild detergent (Brij 96). At low pH fragment A also rapidly and tightly binds to and penetrates model membranes. Binding is reversed when pH is neutralized. The transition pH, the apparent midpoint of the change between the hydrophilic state and the membrane-penetrating hydrophobic state, occurs at about pH 3.5 in the presence of Brij 96 micelles, pH 4 in the presence of small unilamellar vesicles (SUV) composed of zwitterionic phosphatidylcholine, and pH 5 in the presence of SUV composed of 25 mol % anionic phosphatidylglycerol and 75% phosphatidylcholine. The effects of high temperature provide an important clue as to the nature of the changes at low pH. At neutral pH and high temperature, i.e. in the thermally denatured state, a conformational change similar to that observed at low pH occurs, although fragment A does not become hydrophobic. In addition, the effects of low pH and high temperature on the stability of the native state are cumulative. This indicates that the changes in fragment A both at high temperature and at low pH involve denaturation, although there appears to be only partial unfolding under these conditions. Based on the results of this study, the role of fragment A in diphtheria toxin membrane penetration and translocation is evaluated.     

pH-Dependent membrane interactions of diphtheria toxin: A genetic approach

A genetic approach is described for exploring the mechanism by which diphtheria toxin undergoes pH-dependent membrane insertion and transfer of its enzymic A fragment into the cytoplasm of mammalian cells. The cloned toxin expressed inEscherichia coli is secreted to the periplasmic space, where it is processed normally and folds into a native structure. When bacteria synthesizing the toxin are exposed to pH 5, they die rapidly. The toxin undergoes a conformational change that is believed to allow it to be inserted into the bacterial inner membrane and form channels, which proves lethal for the cell. The membrane insertion event mimics the process by which the toxin inserts into the endosomal membrane of mammalian cells, leading to release of the enzymic A fragment into the cytoplasm. The observation of pH-dependent bacterial lethality provides the basis for a positive genetic selection method for mutant forms of the toxin that are altered in ability to undergo membrane insertion or pore formation.     

Virioimmunoassay for studying the interaction of diphtheria toxin with cell membranes]

A model for studying the interaction of diphtheria toxin with cell membranes includes immobilization of purified cell membranes on Sephadex G-25, adsorbtion of toxin on the membranes in the presence of protective colloid, and subsequent detection of adsorbed toxin by means of virioimmunoassay. Diphtheria toxin adsorbed rapidly on membranes of both sensitive (HeLa, macrophages) and resistent to tis action cells, but not on stromaof human erythrocytes. The rate of interaction depends on the concentration of toxin and the temperature of incubation. Adsorbed toxin may be eluted by acidic buffer, 8 M area and 4 M guanidin. HCl, but not by triton X-100, tween 20 and 80, sodium dodecylaulfate and basic buffer.