Membrane Insertion by Anthrax Protective Antigen in Cultured Cells

Qa'Dan, Maen; Christensen, Kenneth A.; Zhang, Lei; Roberts, Thomas M.; Collier, R. John

doi:10.1128/mcb.25.13.5492-5498.2005

Cited by 22 publications

(22 citation statements)

References 33 publications

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“…The insertion half-time was on the order of several seconds, as measured by K ϩ release or enhancement of fluorescence of NBD at position 305 and was much more rapid than that observed with unbound PA. Thus the insertion was largely complete before the complex could proceed down the pathway to inactivation (if such a pathway is in fact operative with membrane-bound PA.) Consistent with observations in other systems, the binding to His-R2 shifted the pH threshold of conversion to the pore to a lower range (9,13,37); mutations in PA known to affect heptamerization or prepore-to-pore conversion inhibited pore formation in the predicted way (3,25); and membrane insertion by a fluorescently tagged form of PA gave results consistent with prior findings in cells and with the ␤-barrel model of pore formation (21). Also, binding of LF N -DTA was shown to depend on proteolytic activation of PA (3).…”

Section: Discussionsupporting

confidence: 87%

“…The intensity of NBD fluorescence at 544 nm increased upon a shift from a polar to a non-polar environment, and earlier we monitored insertion of various residues of the 14-strand ␤-barrel into membranes of Chinese hamster ovary-K1 cells after replacing those residues with Cys and reacting the protein with a thiol-reactive form of NBD (21). In that study, NBD attached to an introduced Cys at position 305 (facing the lipid bilayer) showed a strong increase in fluorescence, whereas at position 306 (facing the aqueous lumen) the fluorophore showed little change.…”

Section: Figure 4 His-r2-dependent Kmentioning

confidence: 98%

“…Time-lapse Intensity Measurements of NBD Emission-PA mutants G305C and N306C were labeled with NBD (N,NЈ-dimethyl-N(iodoacetyl)-NЈ-(7-nitrobenz-2-oxa-1,3-diazol)ethylenediamine) as described (21). The liposomes (DOPC:DOGs-NTA-Ni ϭ 100:8) were incubated with indicated proteins at pH 8.5 for 30 min at room temperature, and the incubated protein-liposome mixture (total volume, 1.8 ml) was transferred to a cuvette with a stirring bar in an ISS K2 fluorometer.…”

Section: Plasmidmentioning

confidence: 99%

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Insertion of Anthrax Protective Antigen into Liposomal Membranes

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Vernier

Wigelsworth

et al. 2007

Journal of Biological Chemistry

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Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K ؉ . Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pHdependent conformational transition to the pore.Many toxins produced by pathogenic bacteria act by enzymically modifying intracellular target molecules. Most of these toxins are bipartite entities, so-called A-B toxins in which one part, the B (binding) moiety, binds to a cell surface receptor and facilitates delivery of the other part, the A (catalytic) moiety, to the cytosol (1). In some of these toxins the B moiety, besides binding to a receptor, also has the ability to undergo membrane insertion and form a pore (channel). The structures and functions of such pores are of great interest in understanding how toxin A moieties translocate across membranes.Anthrax toxin is a tripartite A-B toxin system, composed of two catalytic moieties, edema factor (EF) 2 and lethal factor (LF), and a single receptor-binding/pore-forming moiety, protective antigen (PA). PA (83 kDa) binds to cell-surface receptors and is cleaved by furin or a furin-like protease to an active, 63-kDa form (PA 63 ) (2). PA 63 oligomerizes into a heptameric, receptorbound prepore, which contains high-affinity binding sites for EF and/or LF (3). The entire toxin-receptor complex is internalized by receptor-mediated endocytosis, and within the endosome the prepore undergoes an acidic pH-dependent conformational rearrangement to form a cation-selective, transmembrane pore (4). The PA pore mediates translocation of EF and LF across the endosomal membrane into the cytosol, where EF, an 89-kDa calmodulin-dependent adenylate cyclase, elevates levels of cAMP (5), and LF, a 90-kDa zinc protease, inactivates mitogen-activated proteins kinase kinases ...

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Section: Discussionsupporting

confidence: 87%

Section: Figure 4 His-r2-dependent Kmentioning

confidence: 98%

Section: Plasmidmentioning

confidence: 99%

See 1 more Smart Citation

Insertion of Anthrax Protective Antigen into Liposomal Membranes

Sun

Vernier

Wigelsworth

et al. 2007

Journal of Biological Chemistry

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“…R2 LF N-DTA and prepore were prepared as described in ref. 26. Protein concentrations were determined by spectrophotometry (A 280) and densitometry on SDS/PAGE with BSA as standard.…”

Section: Methodsmentioning

confidence: 99%

Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation

Sun

Lang

Aktories

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The protective antigen (PA) moiety of anthrax toxin forms a heptameric pore in endosomal membranes of mammalian cells and translocates the enzymatic moieties of the toxin to the cytosol of these cells. Phenylalanine-427 (F427), a solvent-exposed residue in the lumen of the pore, was identified earlier as being crucial for the transport function of PA. The seven F427 residues were shown in electrophysiological studies to form a clamp that catalyzes protein translocation through the pore. Here, we demonstrate by a variety of tests that certain F427 mutations also profoundly inhibit the conformational transition of the heptameric PA prepore to the pore and thereby block pore formation in membranes. Lysine, arginine, aspartic acid, or glycine at position 427 strongly inhibited this acidic pH-induced conformational transition, whereas histidine, serine, and threonine had virtually no effect on this step, but inhibited translocation instead. Thus, it is possible to inhibit pore formation or translocation selectively, depending on the choice of the side chain at position 427; and the net inhibition of the PA transport function by any given F427 mutation is the product of its effects on both steps. Mutations inhibiting either or both steps elicited a strong dominant-negative phenotype. These findings demonstrate the dual functions of F427 and underline its central role in transporting the enzymatic moieties of anthrax toxin across membranes.acidic pH ͉ dominant-negative ͉ endosome

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“…The resulting toxic complexes are internalized, and the low pH within the endosome promotes a conformational change in the prepore moiety that allows it to insert into endosomal membranes and form a pore. The conformational transition of the prepore to the pore depends on the association of the 2␤2-2␤3 loops of the seven PA63 subunits to form a membrane-spanning, 14-stranded ␤ barrel (1,21,22).…”

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confidence: 99%

Cross-Linked Forms of the Isolated N-Terminal Domain of the Lethal Factor Are Potent Inhibitors of Anthrax Toxin

et al. 2007

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The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LF N , the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LF N retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20-to 50-fold more potent than monomeric LF N ) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.Bacillus anthracis causes pathology in infected human or animal hosts in part through the concerted action of three proteins, collectively termed anthrax toxin. The toxin consists of two enzymatic moieties, termed lethal factor (LF) and edema factor (EF), and a transport protein, termed protective antigen (PA), that delivers both LF and EF to the cytosol. LF is a 90-kDa zinc-dependent metalloprotease that cleaves mitogen-activated protein kinase kinases (6,20,24), and EF is an 89-kDa calmodulin-dependent adenylate cyclase (12). The intracellular actions of these enzymes impair the functions of various cells and can lead to the death of infected hosts.Delivery of LF and EF to the cytosol begins with binding of PA (83 kDa) to a receptor. Two receptors have been identified: ANTXR1 (for anthrax toxin receptor 1; also known as ATR/ TEM8) and ANTXR2 (for anthrax toxin receptor 2; also known as CMG2) (3, 23). Receptor-bound PA is proteolytically processed by furin or a furin-like protease (19), resulting in the removal of a 20-kDa fragment (PA20) from the N terminus. The remaining, receptor-bound fragment (PA63, 63 kDa) spontaneously oligomerizes, forming a ring-shaped heptamer, called the prepore, which is capable of binding up to three molecules of LF and/or EF with high affinity (17, 18). The resulting toxic complexes are internalized, and the low pH within the endosome promotes a conformational change in the prepore moiety that allows it to insert into endosomal membranes and form a pore. The conformational transition of the prepore to the pore depends on the association of the 2␤2-2␤3

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Membrane Insertion by Anthrax Protective Antigen in Cultured Cells

Cited by 22 publications

References 33 publications

Insertion of Anthrax Protective Antigen into Liposomal Membranes

Insertion of Anthrax Protective Antigen into Liposomal Membranes

Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation

Cross-Linked Forms of the Isolated N-Terminal Domain of the Lethal Factor Are Potent Inhibitors of Anthrax Toxin

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