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. We show using single-channel electrophysiology that PA channels contain two populations of conductance states, which correspond with 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 also report a 3.2-Å crystal structure of the PA octamer. The octamer comprises ∼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.

The protein transporter, anthrax lethal toxin, is comprised of protective antigen (PA), a transmembrane translocase, and lethal factor (LF), a cytotoxic enzyme. Following 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 (LFN). The first α helix and β strand of each LFN 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 on the mechanism of translocation-coupled protein unfolding.

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 to 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 to when these reagents are not present. Protein unfolding induced by chemical and/or thermal denaturation 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.

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 to 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 pre-channel 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.

Energy-consuming nanomachines catalyze the directed movement of biopolymers in the cell. They are found both dissolved in the aqueous cytosol as well as embedded in lipid bilayers. Inquiries into the molecular mechanism of nanomachine-catalyzed biopolymer transport have revealed that these machines are equipped with molecular parts, including adjustable clamps, levers, and adaptors, which interact favorably with substrate polypeptides. Biological nanomachines that catalyze protein transport, known as translocases, often require that their substrate proteins unfold before translocation. An unstructured protein chain is likely entropically challenging to bind, push, or pull in a directional manner, especially in a way that produces an unfolding force. A number of ingenious solutions to this problem are now evident in the anthrax toxin system, a model used to study protein translocation. Here we highlight molecular ratchets and current research on anthrax toxin translocation. A picture is emerging of proton-gradientdriven anthrax toxin translocation, and its associated ratchet mechanism likely applies broadly to other systems. We suggest a cyclical thermodynamic order-to-disorder mechanism (akin to a heatengine cycle) is central to underlying protein translocation: peptide substrates nonspecifically bind to molecular clamps, which possess adjustable affinities; polypeptide substrates compress into helical structures; these clamps undergo proton-gated switching; and the substrate subsequently expands regaining its unfolded state conformational entropy upon translocation.

Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His 6 -XynC, bound to a 220-bp fragment corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the ؊53 and the ؉34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 M XynC. The His 6 -tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (⌬C p ) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system.

Individually, the three protein components of anthrax toxin are nontoxic, but when they assemble, they form active holotoxin complexes. The role of the protective antigen (PA) component of the toxin is to deliver the two other enzyme components, lethal factor (LF) and edema factor (EF), across the plasma membrane and into the cytoplasm of target cells. PA is produced as a proprotein, which must be proteolytically activated, and generally cell-surface activation is mediated by a furin-family protease. Activated PA can then assemble into one of two non-interconverting oligomers, a homoheptamer and homooctamer, which have unique properties. Herein we describe molecular determinants that influence the stoichiometry of PA in toxin complexes. By tethering PA Domain 4 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 Domain 4 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 Domain 4 relative to the main body of PA that favor the formation of either the heptamer or the octamer.

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introductionviaa translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Zplastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Zfixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.