Biology subjects

Iacovache, I.

Publications and source records attributed to Iacovache, I..

4 recordsLinked to original sources

bioRxiv2025

Anthrax toxin receptor 2 is the Receptor for Clostridium perfringens NetF: Structural Insights into Toxin Binding and Pore Formation

Hemolysin {beta}-pore-forming toxins ({beta}PFTs) are key virulence factors of Clostridium perfringens, associated with severe diseases in humans and animals. Yet, the mechanisms by which Clostridium {beta}PFTs recognize and engage specific target cells remain poorly understood. Here, we identify the cellular receptor for C. perfringens necrotizing enteritis toxin F (NetF), a recently discovered toxin implicated in severe enteritis in dogs and foals. We show that NetF binds to the same receptor as anthrax toxin, namely ANTXR2. Using cryo-electron microscopy, we determined the structure of the oligomeric NetF pre-pore as well as the transmembrane pore, both alone and in complex with the extracellular domain of ANTXR2. Unlike anthrax toxin, which binds to the apical MIDAS motif of ANTXR2 - as does the natural ANTXR2 ligand collagen type VI - NetF engages the receptor laterally, spanning both the von Willebrand A and the Ig-like domains. This interaction positions the toxin near the membrane, facilitating contact with membrane lipids and promoting transmembrane pore formation. Our findings uncover key principles of hemolysin {beta}PFT-receptor recognition and advance our understanding of how pathogenic bacteria use these toxins to breach host defenses.

microbiology↗

bioRxiv2024

Genetically Encoded FerriTag as a Specific Label for Cryo-Electron Tomography

Cryo-electron tomography (cryoET) is an important imaging technique that can provide 3D datasets of organelles and proteins at nanometer and sub-nanometer resolution. Recently, combining cryoET with subtomogram averaging has pushed the resolution to 3-4 [A]. However, one main challenge for cryoET is locating target proteins in live cells. Conventional methods such as fluorescent protein tagging and immunogold labeling are not entirely suitable to label small structures in live cells with molecular resolution in vitrified samples. If large proteins, which can be visually identified in cryoET, are directly linked to the target protein, the large tag may alter the target protein structure, localization and function. To address this challenge, we used the rapamycin-induced oligomer formation system, which involves two tags (FKBP and FRB) that can bind together within rapamycin. In our system, the FKBP tag is linked to target protein and the FRB tag is linked to a large protein to create a marker. We chose ferritin as the marker protein because it is a large complex (10-12 nm) and can bind iron to create strong contrast in cryoET. After adding rapamycin to the cell medium, the iron-loaded ferritin accurately indicates the location of the target protein. Recently, in-situ cryoET with subtomogram averaging has been rapidly developing. However, it is still challenging to locate target proteins in live cells, and this method provides a much-needed solution.

cell biology↗

bioRxiv2024

Aerolysin nanopore structure revealed at high resolution in lipid environment

Aerolysin is a {beta}-pore-forming toxin produced by most Aeromonas bacteria which has attracted large attention in the field of nanopore sensing due to its narrow and charged pore lumen. Structurally similar proteins, belonging to the aerolysin-like family, are present throughout all kingdoms of life, but very few of them have been structurally characterized in a lipid environment. Here we present the first high-resolution atomic cryo-EM structures of aerolysin pre-pore and pore in a membrane-like environment. These structures allow the identification of key interactions, which are relevant for the pore formation and for positioning the pore barrel into the membrane with the anchoring {beta}-turn motif now finally observed. Moreover, we elucidate at high resolution the architecture of key mutations and precisely identify four constriction rings in the pore lumen that are highly relevant for nanopore sensing experiments.

biochemistry↗

bioRxiv2021

Cryo-EM structure of the octameric pore of Clostridium perfringens β-toxin

Clostridium perfringens is one of the most widely distributed and successful pathogens. It causes multiple severe diseases in animals and humans and produces an impressive arsenal of toxins with pore-forming properties, most of them belonging to the hemolysin-like family of {beta}-pore forming toxins ({beta}-PFTs). One of the most potent toxins produced by C. perfringens is {beta}-toxin (CPB). This toxin is the main virulence factor of type C strains and essential for the development of a fatal necrotic enteritis in humans and newborn animals. In the present study, we describe the cryo-electron microscopy (cryo-EM) structure of CPB in styrene maleic acid (SMA) discs, which represents the membrane-inserted pore form, at near atomic resolution. We show that CPB forms a homo-oligomeric pore with eightfold symmetry and similar conformation to the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded {beta}-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB defines a new subclass of the hemolysin-like family of {beta}-PFTs s. In addition, we show that the {beta}-barrel protrusion domain can be changed or modified without affecting the pore forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

biophysics↗