Three-dimensional reconstruction of metal replicas of the Helicobacter pylori vacuolating cytotoxin.
The Helicobacter pylori vacuolating cytotoxin (VacA) forms high molecular weight homooligomers which contain either six or seven copies of a 95-kDa polypeptide. Electron microscope visualization of carbon platinum replicas of quick-freeze, deepetched, preparations of VacA has revealed that the oligomers are arranged in flower-like structures with six- or sevenfold radial symmetry, depending on the number of 95-kDa oligomers that they contain. Each monomer is structured in two subunits of 37 and 58 kDa connected by an exposed loop which is a site for proteolytic cleavage. In preparations of VacA which had undergone extensive cleavage at the exposed loop, oligomers of both six- and seven-fold symmetry which appeared flatter were observed; these latter were interpreted as molecules which had lost a complete set of one of the subunits. We exploited a 3D reconstruction of metal replicas of quick-freeze, deep-etched, oligomers, representing the four types of molecules described. All the molecules appear to adhere with the same face toward the mica. Images of rotary shadowed oligomers were processed by multivariate statistical analysis to evidence clusters of equivalent and homogeneous oligomers. 3D reconstructions of the replicas so classified were performed by random conical tilt tomography. In the case of intact molecules (not cleaved) the reconstructions represent both the outer and the inner surfaces of the mold; the latter gives a reasonably accurate sense of the upper surface of the VacA oligomers. These data support the hypothesis that VacA is an AB type toxin and suggest a model in which the smaller of the two subunits is arranged in a uniform ring on the surface of the molecule in such a way as to contribute to the overall stability of the molecule.
Lanzavecchia
S
S
Dipartimento di Chimica Strutturale e Stereochimica Inorganica, Università di Milano, Italy.
Bellon
P L
PL
Lupetti
P
P
Dallai
R
R
Rappuoli
R
R
Telford
J L
JL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D001427:Bacterial Toxins / Q000737:chemistry*; D003603:Cytotoxins / Q000737:chemistry*; D005613:Freeze Etching; D016480:Helicobacter pylori / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D046911:Macromolecular Substances; D011487:Protein Conformation
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D046911:Macromolecular Substances; C074689:VacA protein, Helicobacter pylori