Anthrax Toxin Receptor Drives Protective Antigen Oligomerization and Stabilizes the Heptameric and Octameric Oligomer by a Similar Mechanism

Krantz BA (2010) Anthrax Toxin Receptor Drives Protective Antigen Oligomerization and Stabilizes the Heptameric and Octameric Oligomer by a Similar Mechanism. PLoS ONE 5(11): e13888. doi:10.1371/journal.pone.0013888 Anthrax Toxin Receptor Drives Protective Antigen Oligomerization and Stabilizes the Heptameric and Octameric Oligomer by a Similar Mechanism Alexander F. Kintzer 0 Harry J. Sterling 0 Iok I. Tang 0 Evan R. Williams 0 Bryan A. Krantz 0 Andreas Hofmann, Griffith University, Australia 0 1 Department of Chemistry, University of California, Berkeley, California, United States of America, 2 California Institute for Quantitative Biomedical Research (QB3), University of California, Berkeley, California, United States of America, 3 Department of Molecular and Cell Biology, University of California , Berkeley, California , United States of America Background: Anthrax toxin is comprised of protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins are individually nontoxic; however, when PA assembles with LF and EF, it produces lethal toxin and edema toxin, respectively. Assembly occurs either on cell surfaces or in plasma. In each milieu, PA assembles into a mixture of heptameric and octameric complexes that bind LF and EF. While octameric PA is the predominant form identified in plasma under physiological conditions (pH 7.4, 37uC), heptameric PA is more prevalent on cell surfaces. The difference between these two environments is that the anthrax toxin receptor (ANTXR) binds to PA on cell surfaces. It is known that the extracellular ANTXR domain serves to stabilize toxin complexes containing the PA heptamer by preventing premature PA channel formation-a process that inactivates the toxin. The role of ANTXR in PA oligomerization and in the stabilization of toxin complexes containing octameric PA are not understood. Methodology: Using a fluorescence assembly assay, we show that the extracellular ANTXR domain drives PA oligomerization. Moreover, a dimeric ANTXR construct increases the extent of and accelerates the rate of PA assembly relative to a monomeric ANTXR construct. Mass spectrometry analysis shows that heptameric and octameric PA oligomers bind a full stoichiometric complement of ANTXR domains. Electron microscopy and circular dichroism studies reveal that the two different PA oligomers are equally stabilized by ANTXR interactions. Conclusions: We propose that PA oligomerization is driven by dimeric ANTXR complexes on cell surfaces. Through their interaction with the ANTXR, toxin complexes containing heptameric and octameric PA oligomers are similarly stabilized. Considering both the relative instability of the PA heptamer and extracellular assembly pathway identified in plasma, we propose a means to regulate the development of toxin gradients around sites of infection during anthrax pathogenesis. - Funding: This work was supported by University of California start-up funds and the following National Institutes of Health research grants: R01-AI077703 (to B.A.K.) and R01-GM064712 (to E.R.W.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Anthrax toxin (Atx) [1] is a key virulence factor produced by pathogenic strains of Bacillus anthracis. Atx consists of three nontoxic protein components: protective antigen (PA) is an 83kDa, cell-binding component of Atx that ultimately forms an oligomeric translocase channel, which delivers the two enzyme components, lethal factor (LF) and edema factor (EF), into the cytosol of a host cell [2,3,4]. LF is a 90-kDa, zinc-dependent protease [5,6,7], which cleaves host-cell mitogen-activated protein kinase kinases [5,6]. While PA and LF are individually nontoxic, the combination of LF and PA creates lethal toxin (LT), which can alter cellular physiology and cause death [8]. EF is a 89-kDa, Ca2+/calmodulin-activated adenylyl cyclase [9,10,11]. Analogously, PA and EF combine to form edema toxin (ET), which induces tissue swelling and may also cause death [8,12]. To achieve cytotoxicity, PA, LF, and EF must first self-assemble into holotoxin complexes. There are two different types of assembly pathways: (i) a cell-surface pathway and (ii) a plasmabased/extracellular pathway. In the former mechanism, PA forms complexes on the surface of host cells in a receptor-dependent manner. PA first binds to one of two known Atx receptors (ANTXR): ANTXR1 [13] and ANTXR2 [14]. The PA-ANTXR interaction [15] is stable and dissociates with a half-life measured in days [16]; the interaction involves domains 2 and 4 in PA, such that the latter domain coordinates the receptor Ca2+ or Mg2+ metal ion adhesion site [15,16,17,18]. Receptor-bound PA is then cleaved by a furin-type protease to make the proteolyticallyactivated form, called nPA. After a 20-kDa portion of nPA (PA20) dissociates, the remaining 63-kDa (PA63), receptor-bound portion assembles into a mixture of ring-shaped heptameric (PA7) [17,19,20] and octameric (PA8) [21,22] oligomers. The complexes are endocytosed [23] and brought to an acidic compartment [24]. Under acidic pH conditions, the PA oligomers form translocase channels [25,26], allowing the passage of LF and EF into the cytosol. In a second assembly mechanism, PA, LF, and EF form LT and ET complexes in the blood. In vivo studies of anthrax infection measured high concentrations of toxin components in the blood of infected animals [2,3]. At the later stages of anthrax, PA and LF concentrations reach up to 100 mg/mL and 20 mg/mL, respectively [27]. Analysis of the circulating toxin components revealed that the majority of detectable PA exists as the proteolyticallyprocessed PA63 form, which is either assembled or capable of assembling with LF in a manner analogous to what is observed on cell surfaces [27,28,29]. In vitro bovine-plasma assembly experiments reveal that PA oligomers and LT complexes may form efficiently from full-length PA and LF, where the resulting oligomers contain mixtures of PA7 and PA8 complexes [21,22]. PA7 complexes have a strong propensity for aggregation under physiological conditions (due to their premature conversion to the channel state), suggesting that the toxin requires additional stabilization mechanisms to remain efficacious during infection [21,22,30]. Since PA8 complexes are more stable in plasma under physiological conditions (pH 7.4, 37uC), it has been proposed [22] that the soluble fraction of LT circulating in bloodstream of infected animals [28] may contain an enriched population of the PA8 oligomer. While it is clear that PA8 functions as a stable complex in plasma, it is unknown whether PA7 and PA8 complexes are stabilized differentially on cell surfaces. When the PA heptamer binds to its cellular receptor, ANTXR, the interaction inhibits channel formation, significantly stabilizing PA complexes by , (...truncated)