High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy.
Helicobacter pylori secretes a vacuolating toxin (VacA) that can assemble into water-soluble oligomeric complexes and insert into membranes to form anion-selective channels. Previous studies have described multiple types of oligomeric VacA structures, including single-layered astral arrays, bilayered forms, and two-dimensional crystalline arrays. In the current study, vitrified VacA complexes were examined by cryo-negative staining electron microscopy, views of the different oligomeric structures in multiple orientations were classified and analyzed, and three-dimensional models of the bilayered forms of VacA were constructed with a resolution of about 19 angstroms. These bilayered forms of VacA have a "flower"-like structure, consisting of a central ring surrounded by symmetrically arranged peripheral "petals." Further structural insights were obtained by analyzing a mutant form of VacA (VacADelta6-27), which lacks a unique amino-terminal hydrophobic segment and is defective in the capacity to form membrane channels. Bilayered oligomeric complexes formed by wild-type VacA contained a visible density within the central ring, whereas bilayered complexes formed by VacADelta6-27 lacked this density. These results indicate that deletion of the VacA amino-terminal hydrophobic region causes a structural alteration in the central ring within VacA oligomers, and suggest that the central ring plays an important role in the process by which VacA forms membrane channels.
El-Bez
Catherine
C
Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland. catherine.el-bez@unil.ch
Adrian
Marc
M
Dubochet
Jacques
J
Cover
Timothy L
TL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D016480:Helicobacter pylori / Q000378:metabolism*; D021621:Imaging, Three-Dimensional; D009154:Mutation; D016624:Negative Staining
D001426:Bacterial Proteins; C074689:VacA protein, Helicobacter pylori