Anthrax toxin protective antigen integrates poly-γ-D-glutamate and pH signals to sense the optimal environment for channel formation.

Many toxins assemble into oligomers on the surface of cells. Local chemical cues signal and trigger critical rearrangements of the oligomer, inducing the formation of a membrane-fused or channel state. Bacillus anthracis secretes two virulence factors: a tripartite toxin and a poly-γ-d-glutamic acid capsule (γ-DPGA). The toxin's channel-forming component, protective antigen (PA), oligomerizes to create a prechannel that forms toxic complexes upon binding the two other enzyme components, lethal factor (LF) and edema factor (EF). Following endocytosis into host cells, acidic pH signals the prechannel to form the channel state, which translocates LF and EF into the host cytosol. We report γ-DPGA binds to PA, LF, and EF, exhibiting nanomolar avidity for the PA prechannel oligomer. We show PA channel formation requires the pH-dependent disruption of the intra-PA domain-2-domain-4 (D2-D4) interface. γ-DPGA stabilizes the D2-D4 interface, preventing channel formation both in model membranes and cultured mammalian cells. A 1.9-Šresolution X-ray crystal structure of a D2-D4-interface mutant and corresponding functional studies reveal how stability at the intra-PA interface governs channel formation. We also pinpoint the kinetic pH trigger for channel formation to a residue within PA's membrane-insertion loop at the inter-PA D2-D4 interface. Thus, γ-DPGA may function as a chemical cue, signaling that the local environment is appropriate for toxin assembly but inappropriate for channel formation.

Domain flexibility modulates the heterogeneous assembly mechanism of anthrax toxin protective antigen.

The three protein components of anthrax toxin are nontoxic individually, but they form active holotoxin complexes upon assembly. The role of the protective antigen (PA) component of the toxin is to deliver two other enzyme components, lethal factor and edema factor, across the plasma membrane and into the cytoplasm of target cells. PA is produced as a proprotein, which must be proteolytically activated; generally, cell surface activation is mediated by a furin family protease. Activated PA can then assemble into one of two noninterconverting oligomers, a homoheptamer and a homooctamer, which have unique properties. Herein we describe molecular determinants that influence the stoichiometry of PA in toxin complexes. By tethering PA domain 4 (D4) to domain 2 with two different-length cross-links, we can control the relative proportions of PA heptamers and octamers. The longer cross-link favors octamer formation, whereas the shorter one favors formation of the heptamer. X-ray crystal structures of PA (up to 1.45 Å resolution), including these cross-linked PA constructs, reveal that a hinge-like movement of D4 correlates with the relative preference for each oligomeric architecture. Furthermore, we report the conformation of the flexible loop containing the furin cleavage site and show that, for efficient processing, the furin site cannot be moved ~5 or 6 residues within the loop. We propose that there are different orientations of D4 relative to the main body of PA that favor the formation of either the heptamer or the octamer.

Anthrax toxin receptor drives protective antigen oligomerization and stabilizes the heptameric and octameric oligomer by a similar mechanism.

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, 37°C), 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.

Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers.

The protein transporter anthrax lethal toxin is composed of protective antigen (PA), a transmembrane translocase, and lethal factor (LF), a cytotoxic enzyme. After its assembly into holotoxin complexes, PA forms an oligomeric channel that unfolds LF and translocates it into the host cell. We report the crystal structure of the core of a lethal toxin complex to 3.1-Å resolution; the structure contains a PA octamer bound to four LF PA-binding domains (LF(N)). The first α-helix and ß-strand of each LF(N) unfold and dock into a deep amphipathic cleft on the surface of the PA octamer, which we call the α clamp. The α clamp possesses nonspecific polypeptide binding activity and is functionally relevant to efficient holotoxin assembly, PA octamer formation, and LF unfolding and translocation. This structure provides insight into the mechanism of translocation-coupled protein unfolding.

Role of the protective antigen octamer in the molecular mechanism of anthrax lethal toxin stabilization in plasma.

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-gamma-D-glutamic acid capsule. Atx is comprised of three proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT can assemble on host cell surfaces or extracellularly in plasma. We show that, under physiological conditions in bovine plasma, LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel-forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration, which allows them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood.

The protective antigen component of anthrax toxin forms functional octameric complexes.

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. Using single-channel electrophysiology, we show that PA channels contain two populations of conductance states, which correspond to two different PA pre-channel oligomers observed by electron microscopy-the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here, we report a 3.2-A crystal structure of the PA octamer. The octamer comprises approximately 20-30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus, the 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.