Structure of the Anthrax Protective Antigen D425A Dominant Negative Mutant Reveals a Stalled Intermediate State of Pore Maturation.

The tripartite protein complex produced by anthrax bacteria (Bacillus anthracis) is a member of the AB family of ß-barrel pore-forming toxins. The protective antigen (PA) component forms an oligomeric prepore that assembles on the host cell surface and serves as a scaffold for binding of lethal and edema factors. Following endocytosis, the acidic environment of the late endosome triggers a pH-induced conformational rearrangement to promote maturation of the PA prepore to a functional, membrane spanning pore that facilitates delivery of lethal and edema factors to the cytosol of the infected host. Here, we show that the dominant-negative D425A mutant of PA stalls anthrax pore maturation in an intermediate state at acidic pH. Our 2.7 Å cryo-EM structure of the intermediate state reveals structural rearrangements that involve constriction of the oligomeric pore combined with an intramolecular dissociation of the pore-forming module. In addition to defining the early stages of anthrax pore maturation, the structure identifies asymmetric conformational changes in the oligomeric pore that are influenced by the precise configuration of adjacent protomers.

Site-Specific Labeling and 19F NMR Provide Direct Evidence for Dynamic Behavior of the Anthrax Toxin Pore ϕ-Clamp Structure.

The anthrax toxin protective antigen (PA), the membrane binding and pore-forming component of the anthrax toxin, was studied using 19F NMR. We site-specifically labeled PA with p-fluorophenylalanine (pF-Phe) at Phe427, a critically important residue that comprises the ϕ-clamp that is required for translocation of edema factor (EF) and lethal factor (LF) into the host cell cytosol. We utilized 19F NMR to follow low-pH-induced structural changes in the prepore, alone and bound to the N-terminal PA binding domain of LF, LFN. Our studies indicate that pF-Phe427 is dynamic in the prepore state and then becomes more dynamic in the transition to the pore. An increase in dynamic behavior at the ϕ-clamp may provide the necessary room for movement needed in translocating EF and LF into the cell cytosol.

Binding of the von Willebrand Factor A Domain of Capillary Morphogenesis Protein 2 to Anthrax Protective Antigen Vaccine Reduces Immunogenicity in Mice.

Protective antigen (PA) is a component of anthrax toxin that can elicit toxin-neutralizing antibody responses. PA is also the major antigen in the current vaccine to prevent anthrax, but stability problems with recombinant proteins have complicated the development of new vaccines containing recombinant PA. The relationship between antigen physical stability and immunogenicity is poorly understood, but there are theoretical reasons to think that this parameter can affect immune responses. We investigated the immunogenicity of anthrax PA, in the presence and absence of the soluble von Willebrand factor A domain of the human form of receptor capillary morphogenesis protein 2 (sCMG2), to elicit antibodies to PA in BALB/c mice. Prior studies showed that sCMG2 stabilizes the 83-kDa PA structure to pH, chemical denaturants, temperature, and proteolysis and slows the hydrogen-deuterium exchange rate of histidine residues far from the binding interface. In contrast to a vaccine containing PA without adjuvant, we found that mice immunized with PA in stable complex with sCMG2 showed markedly reduced antibody responses to PA, including toxin-neutralizing antibodies and antibodies to domain 4, which correlated with fewer toxin-neutralizing antibodies. In contrast, mice immunized with PA in concert with a nonbinding mutant of sCMG2 (D50A) showed anti-PA antibody responses similar to those observed with PA alone. Our results suggest that addition of sCMG2 to a PA vaccine formulation is likely to result in a significantly diminished immune response, but we discuss the multitude of factors that could contribute to reduced immunogenicity.IMPORTANCE The anthrax toxin PA is the major immunogen in the current anthrax vaccine (anthrax vaccine adsorbed). Improving the anthrax vaccine for avoidance of a cold chain necessitates improvements in the thermodynamic stability of PA. We address how stabilizing PA using sCMG2 affects PA immunogenicity in BALB/c mice. Although the stability of PA is increased by binding to sCMG2, PA immunogenicity is decreased. This study emphasizes that, while binding of a ligand retains or improves conformational stability without affecting the native sequence, epitope recognition or processing may be affected, abrogating an effective immune response.

Seneca Valley Virus Exploits TEM8, a Collagen Receptor Implicated in Tumor Growth.

Recent studies reveal that Seneca Valley Virus (SVV) exploits tumor endothelial marker 8 (TEM8) for cellular entry, the same surface receptor pirated by bacterial-derived anthrax toxin. This observation is particularly significant as SVV is a known oncolytic virus which selectively infects and kills tumor cells, particularly those of neuroendocrine origin. TEM8 is a transmembrane glycoprotein that is preferentially upregulated in some tumor cell and tumor-associated stromal cell populations. Both TEM8 and SVV have been evaluated for targeting of tumors of multiple origins, but the connection between the two was previously unknown. Here, we review currently understood interactions between TEM8 and SVV, anthrax protective antigen (PA), and collagen VI, a native binding partner of TEM8, with an emphasis on potential therapeutic directions moving forward.

Advances in structure determination by cryo-EM to unravel membrane-spanning pore formation.

The beta pore-forming proteins (ß-PFPs) are a large class of polypeptides that are produced by all Kingdoms of life to contribute to their species' own survival. Pore assembly is a sophisticated multi-step process that includes receptor/membrane recognition and oligomerization events, and is ensued by large-scale structural rearrangements, which facilitate maturation of a prepore into a functional membrane spanning pore. A full understanding of pore formation, assembly, and maturation has traditionally been hindered by a lack of structural data; particularly for assemblies representing differing conformations of functional pores. However, recent advancements in cryo-electron microscopy (cryo-EM) techniques have provided the opportunity to delineate the structures of such flexible complexes, and in different states, to near-atomic resolution. In this review, we place a particular emphasis on the use of cryo-EM to uncover the mechanistic details including architecture, activation, and maturation for some of the prominent members of this family.

The Biophysical Probes 2-fluorohistidine and 4-fluorohistidine: Spectroscopic Signatures and Molecular Properties.

Fluorinated amino acids serve as valuable biological probes, by reporting on local protein structure and dynamics through 19F NMR chemical shifts. 2-fluorohistidine and 4-fluorohistidine, studied here with DFT methods, have even more capabilities for biophysical studies, as their altered pKa values, relative to histidine, allow for studies of the role of proton transfer and tautomeric state in enzymatic mechanisms. Considering the two tautomeric forms of histidine, it was found that 2-fluorohistidine primarily forms the common (for histidine) τ-tautomer at neutral pH, while 4-fluorohistidine exclusively forms the less common π-tautomer. This suggests the two isomers of fluorohistidine can also serve as probes of tautomeric form within biomolecules, both by monitoring NMR chemical shifts and by potential perturbation of the tautomeric equilibrium within biomolecules. Fluorine also enables assignment of tautomeric states in crystal structures. The differences in experimental pKa values between the isomers was found to arise from solvation effects, providing insight into the polarization and molecular properties of each isomer. Results also encompass 13C and 19F NMR chemical shifts, from both tautomers of 2-fluorohistidine and 4-fluorohistidine in a number of different environments. This work can serve as a guide for interpretation of spectroscopic results in biophysical studies employing 2-fluorohistidine and 4-fluorohistidine.

Stability of domain 4 of the anthrax toxin protective antigen and the effect of the VWA domain of CMG2 on stability.

The major immunogenic component of the current anthrax vaccine, anthrax vaccine adsorbed (AVA) is protective antigen (PA). We have shown recently that the thermodynamic stability of PA can be significantly improved by binding to the Von-Willebrand factor A (VWA) domain of capillary morphogenesis protein 2 (CMG2), and improvements in thermodynamic stability may improve storage and long-term stability of PA for use as a vaccine. In order to understand the origin of this increase in stability, we have isolated the receptor binding domain of PA, domain 4 (D4), and have studied the effect of the addition of CMG2 on thermodynamic stability. We are able to determine a binding affinity between D4 and CMG2 (∼300 nM), which is significantly weaker than that between full-length PA and CMG2 (170-300 pM). Unlike full-length PA, we observe very little change in stability of D4 on binding to CMG2, using either fluorescence or 19 F-NMR experiments. Because in previous experiments we could observe a stabilization of both domain 4 and domain 2, the mechanism of stabilization of PA by CMG2 is likely to involve a mutual stabilization of these two domains.

Tautomeric stabilities of 4-fluorohistidine shed new light on mechanistic experiments with labeled ribonuclease A.

Ribonuclease A is the oldest model for studying enzymatic mechanisms, yet questions remain about proton transfer within the active site. Seminal work by Jackson et al. (Science, 1994) labeled Ribonuclease A with 4-fluorohistidine, concluding that active-site histidines act as general acids and bases. Calculations of 4-fluorohistidine indicate that the π-tautomer is predominant in all simulated environments (by ~17 kJ/mol), strongly suggesting that fluoro-labeled ribonuclease A functions with His119 in π-tautomer. The tautomeric form of His119 during proton transfer and tautomerism as a putative mechanistic step in wild-type RNase A remain open questions and should be considered in future mechanistic studies.

Demystifying fluorine chemical shifts: electronic structure calculations address origins of seemingly anomalous (19)F-NMR spectra of fluorohistidine isomers and analogues.

Fluorine NMR spectroscopy is a powerful tool for studying biomolecular structure, dynamics, and ligand binding, yet the origins of (19)F chemical shifts are not well understood. Herein, we use electronic structure calculations to describe the changes in (19)F chemical shifts of 2F- and 4F-histidine/(5-methyl)-imidazole upon acid titration. While the protonation of the 2F species results in a deshielded chemical shift, protonation of the 4F isomer results in an opposite, shielded chemical shift. The deshielding of 2F-histidine/(5-methyl)-imidazole upon protonation can be rationalized by concomitant decreases in charge density on fluorine and a reduced dipole moment. These correlations do not hold for 4F-histidine/(5-methyl)-imidazole, however. Molecular orbital calculations reveal that for the 4F species, there are no lone pair electrons on the fluorine until protonation. Analysis of a series of 4F-imidazole analogues, all with delocalized fluorine electron density, indicates that the deshielding of (19)F chemical shifts through substituent effects correlates with increased C-F bond polarity. In summary, the delocalization of fluorine electrons in the neutral 4F species, with gain of a lone pair upon protonation may help explain the difficulty in developing a predictive framework for fluorine chemical shifts. Ideas debated by chemists over 40 years ago, regarding fluorine's complex electronic effects, are shown to have relevance for understanding and predicting fluorine NMR spectra.

Long-range stabilization of anthrax protective antigen upon binding to CMG2.

Protective antigen (PA) mediates entry of edema factor (EF) and lethal factor (LF) into the cytoplasmic space of the cells through the formation of a membrane-spanning pore. To do this, PA must initially bind to a host cellular receptor. Recent mass spectrometry analysis of PA using histidine hydrogen-deuterium exchange (His-HDX) has shown that binding of the von Willebrand factor A (vWA) domain of the receptor capillary morphogenesis protein-2 (CMG2) lowers the exchange rates of the imidazole C2 hydrogen of several histidines, suggesting that receptor binding decreases the structural flexibility of PA. Here, using His-HDX and fluorescence as a function of denaturant, and protease susceptibility, we show that binding of the vWA domain of CMG2 largely increases the stability of PA and the effect reaches up to 70 Å from the receptor binding interface. We also show that the pKa values and HDX rates of histidines located in separate domains change upon receptor binding. These results indicate that when one end of the protein is anchored, the structure of PA is tightened, noncovalent interactions are strengthened, and the global stability of the protein increases. These findings suggest that CMG2 may be used to stabilize PA in future anthrax vaccines.

(19)F nuclear magnetic resonance and crystallographic studies of 5-fluorotryptophan-labeled anthrax protective antigen and effects of the receptor on stability.

The anthrax protective antigen (PA) is an 83 kDa protein that is one of three protein components of the anthrax toxin, an AB toxin secreted by Bacillus anthracis. PA is capable of undergoing several structural changes, including oligomerization to either a heptameric or octameric structure called the prepore, and at acidic pH a major conformational change to form a membrane-spanning pore. To follow these structural changes at a residue-specific level, we have conducted initial studies in which we have biosynthetically incorporated 5-fluorotryptophan (5-FTrp) into PA, and we have studied the influence of 5-FTrp labeling on the structural stability of PA and on binding to the host receptor capillary morphogenesis protein 2 (CMG2) using (19)F nuclear magnetic resonance (NMR). There are seven tryptophans in PA, but of the four domains in PA, only two contain tryptophans: domain 1 (Trp65, -90, -136, -206, and -226) and domain 2 (Trp346 and -477). Trp346 is of particular interest because of its proximity to the CMG2 binding interface, and because it forms part of the membrane-spanning pore. We show that the (19)F resonance of Trp346 is sensitive to changes in pH, consistent with crystallographic studies, and that receptor binding significantly stabilizes Trp346 to both pH and temperature. In addition, we provide evidence that suggests that resonances from tryptophans distant from the binding interface are also stabilized by the receptor. Our studies highlight the positive impact of receptor binding on protein stability and the use of (19)F NMR in gaining insight into structural changes in a high-molecular weight protein.

Biophysical characterization and immunization studies of dominant negative inhibitor (DNI), a candidate anthrax toxin subunit vaccine.

Dominant Negative Inhibitor (DNI) is a translocation-deficient homolog of recombinant protective antigen of Bacillus anthracis that is a candidate for a next generation anthrax vaccine. This study demonstrates that the biophysical characteristics of the DNI protein stored in lyophilized form at 4°C for 8 y were similar to recombinant Protective Antigen (rPA). To provide information on the accelerated stability of DNI, samples in the lyophilized form were subjected to thermal stress (40°C and 70°C for up to 4 weeks) and thoroughly evaluated using various biophysical and chemical characterization techniques. Results demonstrate preserved structural stability of the DNI protein under extreme conditions, suggesting long-term stability can be achieved for a vaccine that employs DNI, as desired for a biodefense countermeasure. Furthermore, the biological activity of the stressed DNI bound to the adjuvant Alhydrogel (®) was evaluated in mice and it was found that the immunogenicity DNI was not affected by thermal stress.

pH effects on binding between the anthrax protective antigen and the host cellular receptor CMG2.

The anthrax protective antigen (PA) binds to the host cellular receptor capillary morphogenesis protein 2 (CMG2) with high affinity. To gain a better understanding of how pH may affect binding to the receptor, we have investigated the kinetics of binding as a function of pH to the full-length monomeric PA and to two variants: a 2-fluorohistidine-labeled PA (2-FHisPA), which is ∼1 pH unit more stable to variations in pH than WT, and an ∼1 pH unit less stable variant in which Trp346 in the domain 2ß(3) -2ß(4) loop is substituted with a Phe (W346F). We show using stopped-flow fluorescence that the binding rate increases as the pH is lowered for all proteins, with little influence on the rate of dissociation. In addition, we have crystallized PA and the two variants and examine the influence of pH on structure. In contrast to previous X-ray studies, the domain 2ß(3) -2ß(4) loop undergoes little change in structure from pH ∼8 to 5.5 for the WT protein, but for the 2-FHis labeled and W346F mutant there are changes in structure consistent with previous X-ray studies. In accord with pH stability studies, we find that the average B-factor values increase by ∼20-30% for all three proteins at low pH. Our results suggest that for the full-length PA, low pH increases the binding affinity, likely through a change in structure that favors a more "bound-like" conformation.

Comparison of the structural stability and dynamic properties of recombinant anthrax protective antigen and its 2-fluorohistidine-labeled analogue.

Protective antigen (PA) is the primary protein antigenic component of both the currently used anthrax vaccine and related recombinant vaccines under development. An analogue of recombinant PA (2-FHis rPA) has been recently shown to block the key steps of pore formation in the process of inducing cytotoxicity in cells, and thus can potentially be used as an antitoxin or a vaccine. This rPA analogue was produced by fermentation to incorporate the unnatural amino acid 2-fluorohistidine (2-FHis). In this study, the effects of 2-FHis labeling on rPA antigen's conformational stability and dynamic properties were investigated by various biophysical techniques. Temperature/pH stability profiles of rPA and 2-FHis rPA were analyzed by the empirical phase diagram (EPD) approach, and physical stability differences between them were identified. Results showed that rPA and 2-FHis rPA had similar stability at pH 7-8. With decreasing solution pH, however, 2-FHis rPA was found to be more stable. Dynamic sensitive measurements of the two proteins at pH 5 found that 2-FHis rPA was more dynamic and/or differentially hydrated under acidic pH conditions. The biophysical characterization and stability data provide information useful for the potential development of 2-FHis rPA as a more stable rPA vaccine candidate.

Anthrax toxin protective antigen--insights into molecular switching from prepore to pore.

The protective antigen is a key component of the anthrax toxin, as it allows entry of the enzymatic components edema factor and lethal factor into the host cell, through the formation of a membrane spanning pore. This event is absolutely critical for the pathogenesis of anthrax, and although we have yet to understand the mechanism of pore formation, recent developments have provided key insights into how this process may occur. Based on the available data, a model is proposed for the kinetic steps for protective antigen conversion from prepore to pore. In this model, the driving force for pore formation is the formation of the phi (ϕ)-clamp, a region that forms a leak-free seal around the translocating polypeptide. Formation of the ϕ-clamp elicits movements within the prepore that provide steric freedom for the subsequent conformational changes required to form the membrane spanning pore.

Evidence that histidine protonation of receptor-bound anthrax protective antigen is a trigger for pore formation.

The protective antigen (PA) component of the anthrax toxin forms pores within the low pH environment of host endosomes through mechanisms that are poorly understood. It has been proposed that pore formation is dependent on histidine protonation. In previous work, we biosynthetically incorporated 2-fluorohistidine (2-FHis), an isosteric analogue of histidine with a significantly reduced pK(a) ( approximately 1), into PA and showed that the pH-dependent conversion from the soluble prepore to a pore was unchanged. However, we also observed that 2-FHisPA was nonfunctional in the ability to mediate cytotoxicity of CHO-K1 cells by LF(N)-DTA and was defective in translocation through planar lipid bilayers. Here, we show that the defect in cytotoxicity is due to both a defect in translocation and, when bound to the host cellular receptor, an inability to undergo low pH-induced pore formation. Combining X-ray crystallography with hydrogen-deuterium (H-D) exchange mass spectrometry, our studies lead to a model in which hydrogen bonds to the histidine ring are strengthened by receptor binding. The combination of both fluorination and receptor binding is sufficient to block low pH-induced pore formation.

2-Fluoro-l-histidine.

The title compound, C(6)H(8)FN(3)O(2), an analog of histidine, shows a reduced side-chain pK(a) (ca 1). The title structure exhibits a shortening of the bond between the proximal ring N atom and the F-substituted ring C atom, indicating an increase in π-bond character due to an inductive effect of fluorine.

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH.

Domain 4 of the anthrax protective antigen (PA) plays a key role in cellular receptor recognition as well as in pH-dependent pore formation. We present here the 1.95 A crystal structure of domain 4, which adopts a fold that is identical to that observed in the full-length protein. We have also investigated the structural properties of the isolated domain 4 as a function of pH, as well as the pH-dependence on binding to the von Willebrand factor A domain of capillary morphogenesis protein 2 (CMG2). Our results provide evidence that the isolated domain 4 maintains structure and interactions with CMG2 at pH 5, a pH that is known to cause release of the receptor on conversion of the heptameric prepore (PA(63))(7) to a membrane-spanning pore. Our results suggest that receptor release is not driven solely by a pH-induced unfolding of domain 4.

3S-fluoroproline as a probe to monitor proline isomerization during protein folding by 19F-NMR.

Variable-temperature inversion transfer NMR is used to determine the kinetic and thermodynamic parameters of cis-trans isomerization of N-Ac-(3R) and (3S)-fluoroproline-OMe.