Pore-forming toxins (PFT) are water-soluble proteins that possess the remarkable ability to self-assemble on the membrane of target cells, where they form pores causing cell damage. Here, we elucidate the mechanism of action of the haemolytic protein fragaceatoxin C (FraC), a α-barrel PFT, by determining the crystal structures of FraC at four different stages of the lytic mechanism, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore. The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore also exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of a haemolytic toxin.

Pore-forming toxins (PFTs) are proteins that are secreted as soluble molecules and are inserted into membranes to form oligomeric transmembrane pores. In this paper, we report the crystal structure of Fragaceatoxin C (FraC), a PFT isolated from the sea anemone Actinia fragacea, at 1.8 Å resolution. It consists of a crown-shaped nonamer with an external diameter of about 11.0 nm and an internal diameter of approximately 5.0 nm. Cryoelectron microscopy studies of FraC in lipid bilayers reveal the pore structure that traverses the membrane. The shape and dimensions of the crystallographic oligomer are fully consistent with the membrane pore. The FraC structure provides insight into the interactions governing the assembly process and suggests the structural changes that allow for membrane insertion. We propose a nonameric pore model that spans the membrane by forming a lipid-free α-helical bundle pore.

The mechanism by which the HIV-1 MPER epitope is recognized by the potent neutralizing antibody 10E8 at membrane interfaces remains poorly understood. To solve this problem, we have optimized a 10E8 peptide epitope and analyzed the structure and binding activities of the antibody in membrane and membrane-like environments. The X-ray crystal structure of the Fab-peptide complex in detergents revealed for the first time that the epitope of 10E8 comprises a continuous helix spanning the gp41 MPER/transmembrane domain junction (MPER-N-TMD; Env residues 671–687). The MPER-N-TMD helix projects beyond the tip of the heavy-chain complementarity determining region 3 loop, indicating that the antibody sits parallel to the plane of the membrane in binding the native epitope. Biophysical, biochemical and mutational analyses demonstrated that strengthening the affinity of 10E8 for the TMD helix in a membrane environment, correlated with its neutralizing potency. Our research clarifies the molecular mechanisms underlying broad neutralization of HIV-1 by 10E8, and the structure of its natural epitope. The conclusions of our research will guide future vaccine-design strategies targeting MPER.

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

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Background:The structure of the HIV glycoprotein transmembrane anchor is unknown. Results: NMR spectroscopy reveals two helices connected by a flexible segment. The N-terminal helix constitutes a scaffold for neutralizing antibodies. Conclusion:The HIV transmembrane sequence combines two subdomains involved in fusion and immune response modulation during infection. Significance: These data may guide the rational design of vaccines and inhibitors.

Sea anemone actinoporins constitute a protein family of multigene pore-forming toxins (PFT). Equinatoxin II (EqtII), fragaceatoxin C (FraC), and sticholysins I and II (StnI and StnII, respectively), produced by three different sea anemone species, are the only actinoporins whose molecular structures have been studied in depth. These four proteins show high sequence identities and practically coincident three-dimensional structures. However, their pore-forming activity can be quite different depending on the model lipid system employed, a feature that has not been systematically studied before. Therefore, the aim of this work was to evaluate and compare the influence of several distinct membrane conditions on their particular pore-forming behavior. Using a complex model membrane system, such as sheep erythrocytes, StnII showed hemolytic activity much higher than those of the other three actinoporins studied. In lipid model systems, pore-forming ability when assayed against 4:1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin (SM) vesicles, with the membrane binding being the rate-limiting step, decreased in the following order: StnI > StnII > EqtII > FraC. When using 1:1:1 DOPC/SM/cholesterol LUVs, the presence of Chol not only enhanced membrane binding affinities by ∼2 orders of magnitude but also revealed how StnII was much faster than the other three actinoporins in producing calcein release. This ability agrees with the proposal that explains this behavior in terms of their high sequence variability along their first 30 N-terminal residues. The influence of interfacial hydrogen bonding in SM- or dihydro-SM-containing bilayers was also shown to be a generalized feature of the four actinoporins studied. It is finally hypothesized that this observed variable ability could be explained as a consequence of their distinct specificities and/or membrane binding affinities. Eventually, this behavior can be modulated by the nature of their natural target membranes or the interaction with not yet characterized isotoxin forms from the same sea anemone species.

A maltotriose-conjugate can deliver molecules into the cytoplasmic space of Gram-negative bacteria by parasitizing the maltose uptake pathway.