Structural basis for activation of voltage sensor domains in an ion channel TPC1.

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca2+ ions for activation.

On the structure and mechanism of two-pore channels.

In eukaryotes, two-pore channels (TPC1-3) comprise a family of ion channels that regulate the conductance of Na+ and Ca2+ ions across cellular membranes. TPC1-3 form endolysosomal channels, but TPC3 can also function in the plasma membrane. TPC1/3 are voltage-gated channels, but TPC2 opens in response to binding endolysosome-specific lipid phosphatidylinositol-3,5-diphosphate (PI(3,5)P2 ). Filoviruses, such as Ebola, exploit TPC-mediated ion release as a means of escape from the endolysosome during infection. Antagonists that block TPC1/2 channel conductance abrogate filoviral infections. TPC1/2 form complexes with the mechanistic target of rapamycin complex 1 (mTORC1) at the endolysosomal surface that couple cellular metabolic state and cytosolic nutrient concentrations to the control of membrane potential and pH. We determined the X-ray structure of TPC1 from Arabidopsis thaliana (AtTPC1) to 2.87Å resolution-one of the two first reports of a TPC channel structure. Here, we summarize these findings and the implications that the structure may have for understanding endolysosomal control mechanisms and their role in human health.

Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana.

Two-pore channels (TPCs) comprise a subfamily (TPC1-3) of eukaryotic voltage- and ligand-gated cation channels with two non-equivalent tandem pore-forming subunits that dimerize to form quasi-tetramers. Found in vacuolar or endolysosomal membranes, they regulate the conductance of sodium and calcium ions, intravesicular pH, trafficking and excitability. TPCs are activated by a decrease in transmembrane potential and an increase in cytosolic calcium concentrations, are inhibited by low luminal pH and calcium, and are regulated by phosphorylation. Here we report the crystal structure of TPC1 from Arabidopsis thaliana at 2.87 Å resolution as a basis for understanding ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites. We determined sites of phosphorylation in the amino-terminal and carboxy-terminal domains that are positioned to allosterically modulate cytoplasmic Ca(2+) activation. One of the two voltage-sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal Ca(2+) and adopts a conformation distinct from the activated state observed in structures of other voltage-gated ion channels. The structure shows that potent pharmacophore trans-Ned-19 (ref. 17) acts allosterically by clamping the pore domains to VSD2. In animals, Ned-19 prevents infection by Ebola virus and other filoviruses, presumably by altering their fusion with the endolysosome and delivery of their contents into the cytoplasm.

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.

Rapid induction of inflammatory lipid mediators by the inflammasome in vivo.

Detection of microbial products by host inflammasomes is an important mechanism of innate immune surveillance. Inflammasomes activate the caspase-1 (CASP1) protease, which processes the cytokines interleukin (IL)-1ß and IL-18, and initiates a lytic host cell death called pyroptosis. To identify novel CASP1 functions in vivo, we devised a strategy for cytosolic delivery of bacterial flagellin, a specific ligand for the NAIP5 (NLR family, apoptosis inhibitory protein 5)/NLRC4 (NLR family, CARD-domain-containing 4) inflammasome. Here we show that systemic inflammasome activation by flagellin leads to a loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (less than 30 min) death in mice. This unexpected response depends on the inflammasome components NAIP5, NLRC4 and CASP1, but is independent of the production of IL-1ß or IL-18. Instead, inflammasome activation results, within minutes, in an 'eicosanoid storm'--a pathological release of signalling lipids, including prostaglandins and leukotrienes, that rapidly initiate inflammation and vascular fluid loss. Mice deficient in cyclooxygenase-1, a critical enzyme in prostaglandin biosynthesis, are resistant to these rapid pathological effects of systemic inflammasome activation by either flagellin or anthrax lethal toxin. Inflammasome-dependent biosynthesis of eicosanoids is mediated by the activation of cytosolic phospholipase A(2) in resident peritoneal macrophages, which are specifically primed for the production of eicosanoids by high expression of eicosanoid biosynthetic enzymes. Our results therefore identify eicosanoids as a previously unrecognized cell-type-specific signalling output of the inflammasome with marked physiological consequences in vivo.

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.

Supercharging protein complexes from aqueous solution disrupts their native conformations.

The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol (m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

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.

Effects of supercharging reagents on noncovalent complex structure in electrospray ionization from aqueous solutions.

The effects of two supercharging reagents, m-nitrobenzyl alcohol (m-NBA) and sulfolane, on the charge-state distributions and conformations of myoglobin ions formed by electrospray ionization were investigated. Addition of 0.4% m-NBA to aqueous ammonium acetate solutions of myoglobin results in an increase in the maximum charge state from 9+ to 19+, and an increase in the average charge state from 7.9+ to 11.7+, compared with solutions without m-NBA. The extent of supercharging with sulfolane on a per mole basis is lower than that with m-NBA, but comparable charging was obtained at higher concentration. Arrival time distributions obtained from traveling wave ion mobility spectrometry show that the higher charge state ions that are formed with these supercharging reagents are significantly more unfolded than lower charge state ions. Results from circular dichroism spectroscopy show that sulfolane can act as chemical denaturant, destabilizing myoglobin by ∼1.5 kcal/mol/M at 25°C. Because these supercharging reagents have low vapor pressures, aqueous droplets are preferentially enriched in these reagents as evaporation occurs. Less evaporative cooling will occur after the droplets are substantially enriched in the low volatility supercharging reagent, and the droplet temperature should be higher compared with when these reagents are not present. Protein unfolding induced by chemical and/or thermal denaturation in the electrospray droplet appears to be the primary origin of the enhanced charging observed for noncovalent protein complexes formed from aqueous solutions that contain these supercharging reagents, although other factors almost certainly influence the extent of charging as well.

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.