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The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes

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The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes doi:10.1016/j.jmb.2009.07.037 J. Mol. Biol. (2009) 392, 614–629 Available online at www.sciencedirect.com The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes Alexander F. Kintzer1,2, Katie L. Thoren1, Harry J. Sterling1, Ken C. Dong1, Geoffrey K. Feld1, Iok I. Tang1, Teri T. Zhang3, Evan R. Williams1,2, James M. Berger2,3 and Bryan A. Krantz1,2,3⁎ 1Department of Chemistry, The assembly of bacterial toxins and virulence factors is critical to their University of California, function, but the regulation of assembly during infection has not been Berkeley, CA 94720, USA studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring- 2California Institute for shaped homooligomers that bind the two other enzyme components of the Quantitative Biomedical toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To Research (QB3), disrupt the host, these toxic complexes are endocytosed, such that the PA University of California, oligomer forms a membrane-spanning channel that LF and EF translocate Berkeley, CA 94720, USA through to enter the cytosol. Using single-channel electrophysiology, we 3Department of Molecular & show that PA channels contain two populations of conductance states, Cell Biology, University of which correspond to two different PA pre-channel oligomers observed by California, Berkeley, electron microscopy—the well-described heptamer and a novel octamer. CA 94720, USA Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, Received 16 June 2009; dimeric and tetrameric, PA intermediates. Both heptameric and octameric received in revised form PA complexes can translocate LF and EF with similar rates and efficiencies. 11 July 2009; Here, we report a 3.2-Å crystal structure of the PA octamer. The octamer accepted 13 July 2009 comprises ∼20–30% of the oligomers on cells, but outside of the cell, the Available online octamer is more stable than the heptamer under physiological pH. Thus, the 20 July 2009 PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity. © 2009 Elsevier Ltd. All rights reserved. Keywords: anthrax toxin; cell-surface assembly; translocation; lethal toxin; Edited by J. Bowie oligomerization Introduction cell surfaces to make noncovalent, toxic complexes (Supplementary Data Fig. S1). Protective antigen (PA) 1 is the 83 kDa, cell-binding B component that Anthrax toxin (Atx) is a binary toxin (A2B) comprised of three nontoxic proteins that are secreted ultimately forms a translocase channel for the two ∼ — by Bacillus anthracis and combine on eukaryotic host 90 kDa, enzymatically active, A components lethal factor (LF) and edema factor (EF). Following secretion, PA binds to the host cell via one of two *Corresponding author. Department of Chemistry, known Atx receptors, ATR12 and ATR23, and is then University of California, Berkeley, CA 94720, USA. cleaved by a furin-type protease to make the E-mail address: [email protected]. proteolytically activated form, called nPA. The Abbreviations used: Atx, anthrax toxin; PA, protective 63 kDa, receptor-bound portion of nPA then self- antigen; LF, lethal factor; EF, edema factor; WT, wild type; assembles into a ring-shaped homooligomer, or pre- EM, electron microscopy; nanoESI, nanoelectrospray channel, which has been shown to be heptameric.4-8 ionization; MS, mass spectrometry; dsATR, dimeric The pre-channel can bind LF or EF to make lethal or soluble Atx receptor; msATR, monomeric soluble ATR; edema toxins, respectively. These toxin complexes are D4, domain 4; MIL, membrane insertion loop; UBB, endocytosed and brought to an acidic compartment.9 universal bilayer buffer. Under acidic conditions, the PA pre-channel inserts 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved. Protective Antigen Forms Functional Octamers 615 into the membrane to form a translocase channel.10 LF assembly mechanism, since PA oligomers are and EF translocate through the channel to enter the believed to be odd-numbered and heptameric.23,24 cytosol, where they catalyze reactions that disrupt the ATR2 and Atx were recently implicated as factors that host cell (Supplementary Data Fig. S1).11 help the B. anthracis bacterium escape the acidic Analogous to the staphylococcal α-hemolysin phagolysosome following spore germination, sug- pore,12 PA assumes a similar mushroom-shaped gesting Atx components may assemble in hostile architecture8 and β-barrel transmembrane motif.13,14 environments as well.25 Another potentially interest- The β-barrel of the PA channel is similarly narrow ing extracellular assembly mechanism has become (∼15-Å in diameter8,15) but considerably longer apparent in the investigation of animals infected with (∼100 Å) than the α-hemolysin. The narrow channel B. anthracis, where it has been shown that anthrax requires LF and EF to unfold before translocation.16-18 toxin accumulates in the blood of animals.26 Specifi- Acidic endosomal conditions serve two purposes: cally, LF is found alongside proteolytically activated 27,28 first, they destabilize LF and EF by acid nPA, implying that the PA is potentiated for denaturation;18 and second, they drive translocation assembly. Relative to toxin produced in vitro, the toxin via a proton gradient (ΔpH).11 PA also contains a produced in vivo (i.e., isolated from the blood of required ring of phenylalanine side chains, or ϕ animals suffering from anthrax) forms unique assem- clamp, which catalyzes translocation,19 exemplifying blies, as evidenced by their unique resistance to how the structure of the channel is crucial to its antibody binding, and the in vivo-derived toxin is translocase function. more lethal.29 We probe assembly in various cellular Assembly is paramount to Atx function20,21 and its and extracellular contexts using electrophysiology, cellular internalization.22 ATR receptors are interna- electron microscopy, mass spectrometry, and crystal- lized slowly by the host cell, but PA-bound ATR can lography, and we conclude that the activity of the internalize rapidly once it dimerizes,22 making proper toxin may be regulated through assembly, potentially oligomerization a critical step in the internalization affecting the degree of cytotoxicity throughout the pathway. ATR is dimeric, further complicating the stages of anthrax. Fig. 1. Heterogeneous PA chan- nel conductance distributions. (a) Two PA samples were analyzed: (i) PA is nicked by trypsin to make nPA; a20kDapiece(PA20) dissociates, allowing PA to oligomerize into the pre-channel on a Q-Sepharose col- umn, making QPA; and (ii) nPA is mixed with LFN to drive oligomer- ization, making nPA + LFN. Either pre-channel oligomer forms a channel upon inserting into the membrane. (b) Example of 200 Hz-filtered, sin- gle-channel data collected at a Δψ of 20 mV, 100 mM KCl, pH 6.60; γ values computed by γ=i/Δψ are listed next to each channel insertion. (c) Normalized histograms of the estimated single-channel γ values for the QPA and nPA + LFN samples. Data bins are 1 pS wide, and the number of channels, n,ineach sample are normalized for compar- ison. The samples, QPA (n =360; black bars) and nPA + LFN (n=107; red bars), are statistically distinct by a non-parametric, lower-tailed, Whitney-Mann test (p N0.95). (d) Histogram of all the pairwise differ- ences, δ, between measured γ values identified within the same mem- δ brane for the nPA + LFN sample. The histogram was fit to one (dotted line) and two Gaussian (continuous line) functions, δ σ √ π – δ2 σ2 σ √ π δ–μ σ 2 – δ μ σ 2 using A( )=A1/ 1 ( /2) exp( 2 / 1)+A2/ 2 ( /2) [exp(-2(( 2)/ 1) )+exp( 2(( + 2)/ 1) )], with R values of 0.89 and μ 0.96, respectively, yielding best-fit parameters: peak area A1 =470±80, A2 =140±40; mean, 2 =8 (±2) pS; and standard σ σ deviation, 1 =5.5 (±0.5) pS, 2 =9 (±2) pS. (See also Supplementary Data Table S2.) (e) Single-channel current records for smaller (black) and larger (red) PA channels in 100 mM KCl, pH 6.6. Arrows indicate the two respective channel sizes. Data were acquired at 400 Hz and filtered further with a 100 point/σ Gaussian filter to better reveal conductance sub-states. 616 Protective Antigen Forms Functional Octamers Results significantly different (pN0.95). Finally, three separate QPA samples contained a consistently lower mean ∼ conductance of 95 pS relative to two other nPA + LFN PA can form two different channel sizes samples, which had a mean conductance of ∼98 pS. Thus, the method of assembly shifts the single- PA channels were inserted into planar lipid bilayers channel conductance distribution (Fig. 1c). following one of two different assembly methods Inherent variability in bilayer thickness, combined (outlined in Fig. 1a). A pre-oligomerized sample, with the presence of another larger-conductance called QPA (trypsin-nicked PA assembled on Q- conformation, likely contributes to the broad aggre- Sepharose anion-exchange resin10) was applied to gate distributions (Fig. 1c). To circumvent this the bilayer, and discrete single-channel steps were problem, we examined the discrete differences in observed (Fig. 1b). QPA channels had a mean channel conductance within each membrane by conductance of 95.5 pS (n =360). Single-channel tabulating the set of all the pairwise differences (δ) γ γ γ –γ ≠ conductance values ( ) for wild type (WT) PA in values/membrane, { i j}, where i j.Ahisto- – 30 δ channels were reported in the range 85 110 pS. We gram of values recorded for the nPA + LFN sample observed that the QPA sample contained two discrete shows that two sizes of channel conductance are sizes of channel: a prevalent, smaller one and a rarer, present, as the distribution fits best to a two-Gaussian larger one.
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