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.

Scrambling of natural and fluorescently tagged phosphatidylinositol by reconstituted G protein-coupled receptor and TMEM16 scramblases.

Members of the G protein-coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([3H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [3H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [3H]PI hydrolysis increased, indicating that [3H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC-based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase.

Out-of-the-groove transport of lipids by TMEM16 and GPCR scramblases.

Phospholipid scramblases externalize phosphatidylserine to facilitate numerous physiological processes. Several members of the structurally unrelated TMEM16 and G protein-coupled receptor (GPCR) protein families mediate phospholipid scrambling. The structure of a TMEM16 scramblase shows a membrane-exposed hydrophilic cavity, suggesting that scrambling occurs via the ?credit-card" mechanism where lipid headgroups permeate through the cavity while their tails remain associated with the membrane core. Here we show that afTMEM16 and opsin, representatives of the TMEM16 and GCPR scramblase families, transport phospholipids with polyethylene glycol headgroups whose globular dimensions are much larger than the width of the cavity. This suggests that transport of these large headgroups occurs outside rather than within the cavity. These large lipids are scrambled at rates comparable to those of normal phospholipids and their presence in the reconstituted vesicles promotes scrambling of normal phospholipids. This suggests that both large and small phospholipids can move outside the cavity. We propose that the conformational rearrangements underlying TMEM16- and GPCR-mediated credit-card scrambling locally deform the membrane to allow transbilayer lipid translocation outside the cavity and that both mechanisms underlie transport of normal phospholipids.

Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer.

Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca2+-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus Nectria hematococca) is a homodimer with a large cytoplasmic region and a hydrophilic, membrane-exposed groove in each monomer. The groove provides the transbilayer conduit for lipids, but the mechanism by which Ca2+ regulates it is not clear. Because fusion of large protein tags at either the N or C terminus abolishes nhTMEM16 activity, we hypothesized that its cytoplasmic portion containing both termini may regulate lipid translocation via a Ca2+-dependent conformational change. To test this hypothesis, here we used fluorescence methods to map key distances within the nhTMEM16 homodimer and between its termini and the membrane. To this end, we developed functional nhTMEM16 variants bearing an acyl carrier protein (ACP) tag at one or both of the termini. These constructs were fluorescently labeled by ACP synthase-mediated insertion of CoA-conjugated fluorophores and reconstituted into vesicles containing fluorescent lipids to obtain the distance of closest approach between the labeled tag and the membrane via FRET. Fluorescence lifetime measurements with phasor analysis were used to determine the distance between the N and C termini of partnering monomers in the nhTMEM16 homodimer. We now report that the measured distances do not vary significantly between Ca2+-replete and EGTA-treated samples, indicating that whereas the cytoplasmic portion of the protein is important for function, it does not appear to regulate scramblase activity via a detectable conformational change.

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.

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.