The transmembrane domain of diphtheria toxin improves molecular conjugate gene transfer.
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ABSTRACT:
SUBMITTER: Fisher KJ
PROVIDER: S-EPMC1218035 | biostudies-other | 1997 Jan
REPOSITORIES: biostudies-other
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Project description:A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by blast, clustal w, and meme computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB(389)IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102/6TG cells that express the T1 peptide, Hut102/6TG-T1. In contrast to the parental cell line, Hut102/6TG-T1 cells are ca. 10(4)-fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102/6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit beta to this region of the diphtheria toxin transmembrane domain.
| S-EPMC1257389 | 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.
| S-EPMC4113555 | biostudies-literature
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 diphtheria toxin T domain helps translocate the A chain of the toxin across membranes. To gain insight into translocation, the membrane topography of key residues in T domain attached to the A chain (AT protein) was compared to that in the isolated T domain using fluorescence techniques. This study demonstrates that residues in T domain hydrophobic helices (TH5-TH9) tended to be less exposed to aqueous solution in the AT protein than in the isolated T domain. Under conditions in which the loop connecting TH5 to TH6/7 is located stably on the cis (insertion) side of the membrane in the isolated T domain, it moves between the cis and trans sides of the membrane in the AT protein. This is indicative of the formation of a dynamic, transient transmembrane hairpin topography by TH5-TH7 in the AT protein. Since TH8 and TH9 also form a transmembrane hairpin, this means that TH5-TH9 may form a cluster of transmembrane helices. These helices have a nonpolar surface likely to face the lipid bilayer in a helix cluster and a surface rich in uncharged hydrophilic residues which in a helix cluster would likely be facing inward (and perhaps be pore-lining). This uncharged hydrophilic surface could play a crucial role in translocation, interacting transiently with the translocating A chain. A similar motif can be found in, and may be important for, other protein translocation systems.
| S-EPMC2783497 | biostudies-literature
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.
| S-EPMC4385515 | biostudies-literature
Project description:Hepatocellular carcinoma (HCC) is a major global malignancy, responsible for >90% of primary liver cancers. Currently available therapeutic options have poor performances due to the highly heterogeneous nature of the tumor cells; recurrence is highly probable, and some patients develop resistances to the therapies. Accordingly, the development of a novel therapy is essential. We assessed gene therapy for HCC using a diphtheria toxin fragment A (DTA) gene-expressing plasmid, utilizing a non-viral hydrodynamics-based procedure. The antitumor effect of DTA expression in HCC cell lines (and alpha-fetoprotein (AFP) promoter selectivity) is assessed in vitro by examining HCC cell growth. Moreover, the effect and safety of the AFP promoter-selective DTA expression was examined in vivo using an HCC mice model established by the hydrodynamic gene delivery of the yes-associated protein (YAP)-expressing plasmid. The protein synthesis in DTA transfected cells is inhibited by the disappearance of tdTomato and GFP expression co-transfected upon the delivery of the DTA plasmid; the HCC cell growth is inhibited by the expression of DTA in HCC cells in an AFP promoter-selective manner. A significant inhibition of HCC occurrence and the suppression of the tumor marker of AFP and des-gamma-carboxy prothrombin can be seen in mice groups treated with hydrodynamic gene delivery of DTA, both 0 and 2 months after the YAP gene delivery. These results suggest that DTA gene therapy is effective for HCC.
| S-EPMC7072394 | biostudies-literature
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.
| S-EPMC4259893 | biostudies-literature
Project description:Diphtheria toxin translocation (T) domain undergoes conformational changes in acidic solution and associates with the lipid membranes, followed by refolding and transmembrane insertion of two nonpolar helices. This process is an essential step in delivery of the toxic catalytic domain of the diphtheria toxin to the infected cell, yet its molecular determinants are poorly characterized and understood. Therefore, an atomistic model of the T-domain-membrane interaction is needed to help characterize factors responsible for such association. In this work, we present atomistic model structures of T-domain membrane-bound conformations and investigate structural factors responsible for T-domain affinity with the lipid bilayer in acidic solution using all-atom molecular dynamics (MD) simulations. The initial models of the protein conformations and protein-membrane association that serve as starting points in the present work were developed using atomistic simulations of partial unfolding of the T-domain in acidic solution (Kurnikov, I. V.; et al. J. Mol. Biol. 2013, 425, 2752-2764), and coarse-grained simulations of the T-domain association with the membranes of various compositions (Flores-Canales, J. C.; et al. J. Membr. Biol. 2015, 248, 529-543). In this work we present atomistic level modeling of two distinct configurations of the T-domain in association with the anionic lipid bilayer. In microsecond-long MD simulations both conformations retain their compact structure and gradually penetrate deeper into the bilayer interface. One membrane-bound conformation is stabilized by the protein contacts with the lipid hydrophobic core. The second modeled conformation is initially inserted less deeply and forms multiple contacts with the lipid at the interface (headgroup) region. Such contacts are formed by the charged and hydrophilic groups of partially unfolded terminal helixes and loops. Neutralization of the acidic residues at the membrane interface allows for deeper insertion of the protein and reorientation of the protein at the membrane interface, which corroborates that acidic residue protonation as well as presence of the anionic lipids may play a role in the membrane association and further membrane insertion of the T-domain as implicated in experiments. All simulations reported in this work were performed using AMBER force-field on Anton supercomputer. To perform these reported simulations, we developed and carefully tested a force-field for the anionic 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (POPG) lipid, compatible with the Amber 99SB force-field and stable in microsecond-long MD simulations in isothermal-isobaric ensemble.
| S-EPMC4916650 | biostudies-literature
Project description:Protein-side-chain protonation, coupled to conformational rearrangements, is one way of regulating physiological function caused by changes in protein environment. Specifically, protonation of histidine residues has been implicated in pH-dependent conformational switching in several systems, including the diphtheria toxin translocation (T) domain, which is responsible for the toxin's cellular entry via the endosomal pathway. Our previous studies a) identified protonation of H257 as a major component of the T domain's conformational switch and b) suggested the possibility of a neighboring H223 acting as a modulator, affecting the protonation of H257 and preventing premature conformational changes outside the endosome. To verify this "safety-latch" hypothesis, we report here the pH-dependent folding and membrane interactions of the T domain of the wild-type and that of the H223Q mutant, which lacks the latch. Thermal unfolding of the T domain, measured by circular dichroism, revealed that the reduction in the transition temperature for helical unfolding for an H223Q mutant starts at less acidic conditions (pH <7.5) relative to the wild-type protein (pH <6.5). Hydrogen-deuterium-exchange mass spectrometry demonstrates that the H223Q replacement results in a loss of stability of the amphipathic helices TH1-3 and the hydrophobic core helix TH8 at pH 6.5. That this destabilization occurs in solution correlates well with the pH-range shift for the onset of the membrane permeabilization and translocation activity of the T domain, confirming our initial hypothesis that H223 protonation guards against early refolding. Taken together, these results demonstrate that histidine protonation can fine-tune pH-dependent switching in physiologically relevant systems.
| S-EPMC5103022 | biostudies-literature