Structure determination of two pore forming toxins by cryo-negative staining and single particle three-Dimensional reconstruction

Abtract: Cryo-electron microscopy of biological unordered and non aggregated molecules is a powerful approach for macromolecular assemblies, which are often too large and flexible to be studied by X-ray crystallography. Low temperature that preserves the native hydrated macromolecular structure confers to this technique the power to study conformational changes. Electron micrographs are very noisy and show low intrinsic contrast. Although there is no limits to the mass of the particles that can be analysed, experience place a lower size cutoff of ~250 kDa molecular weight. In order to go beyond these limits, the cryo-negative staining has been developped in our laboratory. The presence of a stain (molybdate amonium) makes possible to vizualize the particles with a enhanced SNR while keeping the specimen in a good state of preservation and reducing electron-beam sensity of vitrified specimen. In the present work, this method allowed us the study of two different pore forming toxins (PFTs), the Helicobacter Pylori vacuolating toxin,'VacA' unrelated to any other proteins, and a mutant form of the Aeromonas Hydrophila toxin, aerolysin (Y221G). Image analysis and 3D reconstruction of the wild type toxin VacA revealed that the monomer assembled into a number of different oligomers, all of which keeping elements of remarkable mobility. The structural comparaison of the wild type form of VacA and a mutant form where a unique hydrophobic region located near its amino terminus was deleted helped us to localize this hydrophobic domain whithin the VacA oligomeric structure and to provide insight into how this domain contributes to the formation of membrane channels by VacA. A high resolution model of a mutant form of aerolysin (Y221G), where a single point mutation has converted a normally membrane-embeded toxin into a soluble complex was obtained by the combination of X-ray data with the electron microscopy map. This model helped us to understand the role of the tyrosine 221 in the exposition of the hydrophobic regions that form the membrane- inserted channel. Finally, the small protein (~200 kDa) dipeptidyl-peptidase IV was also any studied by this technique. Resume La cryo-microscopie electronique des molecules biologiques desordonnees et desagregees est une approche puissante pour l'etude structurale d'assemblages macromoleculaires trop volumineux pour etre etudies par cristallographie aux rayons X. La basse temperature a laquelle les specimens sont observes preserve les macromolecules dans leur etat natif et hydrate et confere a cette technique le pouvoir d'etudier les differents etats conformationnels. Les images de microscopie electronique obtenues, sont bruitees et presentent un contraste faible limitant ainsi l'observation de particules de faible poids moleculaire d'environ 250 kDa. Afin de surpasser ces limites, la cryo-coloration negative a ete developpee dans notre laboratoire. La presence d'un colorant (le molybdate d'ammonium) permet la visualisation des particules avec un rapport signal-sur-bruit augmente tout en preservant le specimen et en le protegeant des degâts du faisceau electronique. Dans ce travail, cette methode nous a permis d'etudier deux toxines bacteriennes differentes formant des pores membranaires, la toxine VacA d'Helicobacter pyiori apparentee a aucune autre toxine connue et une forme mutante de la toxine aerolysine d'Aeromonas Hydrophila (Y221G). Le traitement d'images et la reconstruction tri-dimensionnelle de la toxine VacA de type sauvage a permis de mettre en lumiere l'assemblage des monomeres en oligomeres tout en conservant des elements d'une remarquable mobilite. La structure de la forme mutante de VacA pour laquelle l'unique region hydrophobe a ete tronquee est comparee avec celle de la forme de type sauvage Cette comparaison a mis en evidence la localisation de ce domaine hydrophobe a l'interieur de la structure des oligomeres ainsi que la contribution de ce domaine dans la formation de canaux membranaires par VacA. La toxine aerolysine (Y221G) est le resultat d'une mutation unique qui permet la conversion d'une toxine inseree dans une membrane en un complexe soluble. Un modele a l'echelle atomique a ete obtenu pour ce mutant en combinant la carte de microscopie electronique tri-dimensionnelle aux donnees de cristallographie aux rayons X. Ce modele souligne le role de la tyrosine 221 dans l'exposition des regions hydrophobes qui forment le canal membranaire. Enfin, la derniere proteine etudiee, la dipeptidyl-peptidase (DPPIV) (~200 kDa) a demontre que la cryo-coloration negative permet l'etude structurale de proteines plus petites que celles qui ont ete d'etudiees jusqu'ici.