[show abstract][hide abstract] ABSTRACT: Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.
[show abstract][hide abstract] ABSTRACT: Chimeric protein toxins that act selectively on cells expressing a designated receptor may serve as investigational probes and/or antitumor agents. Here we report use of the enzyme sortase A (SrtA) to create 4 chimeric toxins designed to selectively kill cells bearing the tumor marker, HER2. We first expressed and purified: (i) a receptor recognition-deficient form of diphtheria toxin (DT) that lacks its receptor binding domain and (ii) a mutated, receptor-binding-deficient form of anthrax protective antigen (PA). Both proteins carried at the C terminus the sortase recognition sequence LPETGG and a H6 affinity tag. Each toxin protein was mixed with SrtA plus either of two HER2-recognition proteins-a single-chain antibody fragment or an Affibody-both carrying an N terminal G5 tag. With wild-type SrtA the fusion reaction between the toxin and receptor-recognition proteins approached completion only after several hours, whereas with an evolved form of the enzyme, SrtA*, the reaction was virtually complete within 5 minutes. The four fusion toxins were purified and shown to kill HER2-positive cells in culture with high specificity. Sortase-mediated ligation of binary combinations of diverse natively folded proteins offers a facile way to produce large sets of chimeric proteins for research and medicine.
Molecular Cancer Therapeutics 08/2013; · 5.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have visualized by cryo-electron microscopy (cryo-EM) the complex of the anthrax protective antigen (PA) translocon and the N-terminal domain of anthrax lethal factor (LFN ) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16Å by single-particle analysis and three-dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LFN , though not distinguished due to the seven-fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LFN extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM-defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.
[show abstract][hide abstract] ABSTRACT: We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LF(N) ) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (∼43%). Three-dimensional analysis of negatively stained single particles revealed the LF(N) -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF(N) molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LF(N) -PA nanodisc structures and use of advanced automated particle selection methods.
[show abstract][hide abstract] ABSTRACT: Studying how pathogens subvert the host to cause disease has contributed to the understanding of fundamental cell biology. Bacillus anthracis, the causative agent of anthrax, produces the virulence factor lethal toxin to disarm host immunity and cause pathology. We conducted a phenotypic small molecule screen to identify inhibitors of lethal toxin-induced macrophage cell death and used an ordered series of secondary assays to characterize the hits and determine their effects on cellular function. We identified a structurally diverse set of small molecules that act at various points along the lethal toxin pathway, including inhibitors of endocytosis; natural product inhibitors of organelle acidification (e.g. the botulinum neurotoxin inhibitor, toosendanin); and a novel proteasome inhibitor, 4MNB (4-methoxy-2-[2-(5-methoxy-2-nitrosophenyl)ethyl]-1-nitrosobenzene). Many of the compounds, including three drugs approved for use in humans, also protected against the related Clostridium difficile toxin TcdB, further demonstrating their value as novel tools for perturbation and study of toxin biology and host cellular processes, and highlighting potential new strategies for intervening on toxin-mediated diseases.
ACS Chemical Biology 01/2013; · 5.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: Targeted therapeutics have emerged in recent years as an attractive approach to treating various types of cancer. One approach is to modify a cytocidal protein toxin to direct its action to a specific population of cancer cells. We created a targeted toxin in which the receptor-binding and pore-forming moiety of anthrax toxin, termed Protective Antigen (PA), was modified to redirect its receptor specificity to HER2, a marker expressed at the surface of a significant fraction of breast and ovarian tumors. The resulting fusion protein (mPA-ZHER2) delivered cytocidal effectors specifically into HER2-positive tumor cells, including a trastuzumab-resistant line, causing death of the cells. No off-target killing of HER2-negative cells was observed, either with homogeneous populations or with mixtures of HER2-positive and HER2-negative cells. A mixture of mPA variants targeting different receptors mediated killing of cells bearing either receptor, without affecting cells devoid of these receptors. Anthrax toxin may serve as an effective platform for developing therapeutics to ablate cells bearing HER2 or other tumor-specific cell-surface markers.
[show abstract][hide abstract] ABSTRACT: The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes which take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here, we report that C-terminal histidine residues H322, H323 and H372 of the isolated T-domain are important for the effective transition from the inserted intermediate to the functional open-channel state in the insertion/translocation pathway. We have mutated these histidine residues into triple-Q, double-Q and single-Q mutants and followed their behavior along the insertion/translocation pathway by fluorescence and CD spectroscopy and functional assays in membranes. Triple, double and single mutations caused a loss of characteristic conductance in planar bilayers in different degrees, as well as perturbation in the fold of the inserted state of T-domain relative to the WT T-domain. Interestingly, none of the mutations displayed appreciable alterations in the folding in solution or in the ability to destabilize vesicles to cause leakage of preloaded fluorescent markers. In addition, we have found that a triple mutation of these residues into glutamine or arginine prevents the effective translocation of the N-terminus. Thus, we suggest that C-terminal histidine residues play an important role in the formation of the final and functional inserted state fold of T-domain.
[show abstract][hide abstract] ABSTRACT: The function of diphtheria toxin translocation (T) domain is a transfer of catalytic domain across endosomal membrane in response to acidification. The goal of this study is to develop a simple and reliable in vitro functional assay for the T-domain, one that can be used to investigate structure-function relationships using interactions of various mutants with lipid bilayers of various compositions. Traditionally the activity of the T-domain is estimated either by measuring conductance in planar lipid bilayers or by measuring the release of fluorescent markers from large unilamellar lipid vesicles. We have applied these measurements to a series of T-domain mutants with substitutions in His residues involved in modulating pH-dependent refolding and insertion of the protein. The comparison of the results with those obtained in cell cytotoxicity assay performed with full length toxin revealed lack of correlation of the leakage-based assay with other assays. Our data suggest that release of fluorescent markers is related to the stress induced by the interfacial binding, rather than by the insertion of the protein in a functional conformation. To resolve the contradiction between cellular and in vitro measurements we have developed a translocation test based on the cleavage of the N-terminal part of T-domain upon its translocation into thrombin-loaded vesicles. Application of this test to a series of mutants correlated well with results of cytotoxicity. In the other set of experiments we have investigated lipid dependence - non-linear changes with surface potential (which is in contrast with leakage measurements) confirming our previous suggestion that formation of the final inserted state is modulated by anionic lipids. NIH GM069783 (ASL), GM-29210 (AF), AI-022021(RGC) and Fulbright Foundation.
[show abstract][hide abstract] ABSTRACT: The actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo. IMPORTANCE: Bacterial toxins that act within mammalian cells have receptor-dependent mechanisms to transport their enzymatic components to the cytoplasmic compartment. By inactivating or otherwise modifying their respective intracellular targets, these intracellular effectors disrupt metabolic pathways and in some cases cause death of the cell. Our results show that the receptor specificity of the transport protein of anthrax toxin may be readily changed, raising the possibility that receptor-redirected forms of protective antigen (PA) and PA homologs may be useful for research and medical applications requiring modification or ablation of designated populations of cells.
[show abstract][hide abstract] ABSTRACT: The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here we report that replacement of the three C-terminal histidine residues, H322, H323, and H372, in triple-R or triple-Q mutants prevents effective translocation of the N-terminus. Introduction of these mutations in the full-length toxin results in decrease of its potency. In the context of isolated T-domain, these mutations cause loss of characteristic conductance in planar bilayers. Surprisingly, these mutations do not affect general folding in solution, protein interaction with the membranes, insertion of the consensus transmembrane helical hairpin TH8-9, or the ability of the T-domain to destabilize vesicles to cause leakage of fluorescent markers. Thus, the C-terminal histidine residues are critical for the transition from the inserted intermediate state to the open-channel state in the insertion/translocation pathway of the T-domain.
[show abstract][hide abstract] ABSTRACT: Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.
Proceedings of the National Academy of Sciences 09/2011; 108(40):16577-81. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Electrophysiological studies of wild-type and mutated forms of anthrax protective antigen (PA) suggest that the Phe clamp, a structure formed by the Phe427 residues within the lumen of the oligomeric PA pore, binds the unstructured N-terminus of the lethal factor and the edema factor during initiation of translocation. We now show by electrophysiological measurements and gel shift assays that a single Cys introduced into the Phe clamp can form a disulfide bond with a Cys placed at the N-terminus of the isolated N-terminal domain of LF. These results demonstrate direct contact of these Cys residues, supporting a model in which the interaction of the unstructured N-terminus of the translocated moieties with the Phe clamp initiates N- to C-terminal threading of these moieties through the pore.
[show abstract][hide abstract] ABSTRACT: The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores that translocate the enzymatic moieties of the toxin--lethal factor (LF) and edema factor (EF)--across the endosomal membrane of mammalian cells. Here we describe site-directed spin-labeling studies that identify interactions of LF with the prepore and pore conformations of PA. Our results reveal a direct interaction between the extreme N terminus of LF (residues 2-5) and the Φ-clamp, a structure within the lumen of the pore that catalyzes translocation. Also, consistent with a recent crystallographic model, we find that, upon binding of the translocation substrate to PA, LF helix α1 separates from helices α2 and α3 and binds in the α-clamp of PA. These interactions, together with the binding of the globular part of the N-terminal domain of LF to domain 1' of PA, indicate that LF interacts with the PA pore at three distinct sites. Our findings elucidate the state from which translocation of LF and EF proceeds through the PA pore.
Proceedings of the National Academy of Sciences 02/2011; 108(5):1868-73. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LF(N)DTA. LF(N) mediates uptake of LF(N)DTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP-ribosylates elongation factor-2, allowing for detection of small amounts of translocated LF(N)DTA. Cyclosporin A, which inhibits peptidyl-prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LF(N)DTA into the cytosol of CHO-K1 cells and protected cells from intoxication by LF(N)DTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LF(N)DTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LF(N)DTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LF(N)DTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Molecular Biology 09/2010; 402(1):1-7. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the cell entry and intracellular trafficking of anthrax oedema factor (EF) and lethal factor (LF), they were C-terminally fused to the enhanced green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent proteins. Both chimeras bound to the surface of BHK cells treated with protective antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and the two anthrax factors were found to traffic along the same endocytic route and with identical kinetics, indicating that their intracellular path is essentially dictated by PA. Colocalization studies indicated that anthrax toxins enter caveolin-1 containing compartments and then endosomes marked by phoshatidylinositol 3-phoshate and Rab5, but not by early endosome antigen 1 and transferrin. After 40 min, both EF and LF chimeras were observed to localize within late compartments. Eventually, LF and EF appeared in the cytosol with a time-course consistent with translocation from late endosomes. Only the EGFP derivatives reached the cytosol because they are translocated by the PA channel, while the mCherry derivatives are not. This difference is attributed to a higher resistance of mCherry to unfolding. After translocation, LF disperses in the cytosol, while EF localizes on the cytosolic face of late endosomes.
[show abstract][hide abstract] ABSTRACT: Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >or=1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.
Journal of Biological Chemistry 03/2010; 285(11):8130-7. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-A resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.
Proceedings of the National Academy of Sciences 02/2010; 107(8):3453-7. · 9.74 Impact Factor