Subcloning and characterization of the binding domain of fragment B of diphtheria toxin.
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ABSTRACT:
SUBMITTER: Esbensen QY
PROVIDER: S-EPMC1134513 | biostudies-other | 1993 Sep
REPOSITORIES: biostudies-other
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Project description:The diphtheria toxin T domain translocates the catalytic C domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited wild-type-like activity in channel formation, N-terminus translocation, and cytotoxicity assays. Moreover, membrane permeabilization caused by the H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.
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Project description:Endosomal cargo travels through a dynamic vesicle network en route to degradation by lysosomes or recycling through the Golgi apparatus back to the cell surface. Rab5 is a key determinant of the early endosomes by organizing effector proteins in specific subdomains and mediating early endosome fusion. We find that early endosome morphogenesis and maturation is disrupted by diphtheria toxin (DT). Rab5 bound endosomes increase in size and in Rab5 content due to luminal toxin exposure, whereas Rab7 positive endosomes are not detectably altered. These changes appear to be caused by an activity of the toxin entry domain (T domain) as mutations inactivating either the receptor binding (CRM107) or ADP-ribosyl transferase (CRM197) activities do not inhibit the effect of DT on endosome morphogenesis. In contrast, mutations in the T domain or diminishing the endosomal pH gradient, which prevents T domain membrane insertion, inhibit these endosome changes. The change in size appears to be due to changing the early endosome fission-fusion equilibrium. The Rab5 membrane exchange rate, assessed with photoactivatable GFP-Rab5, decreases in the presence of DT. These changes to endosomes may reflect activities of the T domain that mediate toxin entry to the cytosol. The nontoxic mutant DT, CRM197, yields a new tool to manipulate endosome dynamics in living cells.
| S-EPMC2430352 | biostudies-literature
Project description:The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild-type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.
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Project description:Natural diphtheria toxin is synthesized as a single polypeptide chain that is activated by cleavage into an A- and a B-fragment, which are linked by a disulphide bond. In the present work the ability of independently translated A- and B-fragments to associate was investigated. Low amounts of A- and B-fragments synthesized in vitro were mixed under conditions that allowed formation of a disulphide bridge between the fragments. Under these conditions toxin was reconstituted in close to 100% yield and found to be as toxic to Vero cells as natural diphtheria toxin. Efficient association between the A- and B-fragment was dependent on the formation of a disulphide bridge. Reconstituted toxin obtained from one [35S]methionine-labelled fragment and one unlabelled fragment proved useful in translocation studies. Addition of a number of different polypeptides to the N- and C-termini of either fragment did not, in most cases, prevent reconstitution. The ready reconstitution allows easy manipulations with the toxin to form targeted molecules and to develop diphtheria toxin as a vector for translocation of peptides to the cytosol. The fact that the reconstituted toxin does not need to be nicked with proteinases to be active allows experimentation with proteinase-sensitive constructs.
| S-EPMC1130734 | biostudies-other
Project description:1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH(2), NAD, NADPH(2) and NADP. 2. The binding stoicheiometry is 2moles of NADH(2) and 1mole of NADPH(2)/mole of diphtheria toxin. The binding sites for NADH(2) appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0.01I, pH7 and 25 degrees are 0.7x10(-6)m for NADH(2) and 0.45x10(-6)m for NADPH(2). Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH(2) and NADPH(2) may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH(2) are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin-nucleotide complexes is discussed in this regard.
| S-EPMC1198356 | biostudies-other
Project description:The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism, and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3-5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately -8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between -9.5 and -12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain.
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Project description:Vectors based on the formation of a soluble DNA-polycation complex are being developed for the treatment of human diseases. These complexes are rapidly taken up by receptor-mediated endocytosis, but are inefficiently delivered to the nucleus owing to entrapment in membrane-bound vesicles. In this study we introduced the transmembrane domain of diphtheria toxin into a DNA-polycation conjugate complex in an effort to increase gene transfer by membrane perturbation. The transmembrane domain of diphtheria toxin was expressed in Escherichia coli as a maltose-binding protein fusion and chemically coupled to high-molecular-mass poly-L-lysine. Incorporation of this conjugate into a traditional complex formed with a luciferase-containing plasmid with an asialo-orosomucoid-polycation conjugate significantly increased transfection efficiency in vitro in a manner proportional to the amount of diphtheria toxin incorporated. The delivery of luciferase RNA transcript was similarly increased when complexed with similar polycation conjugates. This study uses the structural biology of a bacterial protein to improve polycation-based gene delivery.
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Project description:The function of diphtheria toxin translocation (T) domain is to transfer the catalytic domain across the endosomal membrane upon acidification. The goal of this study was to develop and apply an in vitro functional assay for T domain activity, suitable for investigation of structure-function relationships of translocation across lipid bilayers of various compositions. Traditionally, T domain activity in vitro is estimated by measuring either conductance in planar lipid bilayers or the release of fluorescent markers from lipid vesicles. While an in vivo cell death assay is the most relevant to physiological function, it cannot be applied to studying the effects of pH or membrane lipid composition on translocation. Here we suggest an assay based on cleavage of the N-terminal part of T domain upon translocation into protease-loaded vesicles. A series of control experiment was used to confirm that cleavage occurs inside the vesicle and not as the result of vesicle disruption. Translocation of the N-terminus of the T domain is shown to require the presence of a critical fraction of anionic lipids, which is consistent with our previous biophysical measurements of insertion. Application of the proposed assay to a series of T domain mutants correlated well with the results of cytotoxicity assay.
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Project description:The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five beta-strands and three helices arranged into a partially orthogonal, two-sheet beta-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in beta-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.
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