3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.
Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.
Reyrat
J M
JM
Chiron S.p.A., IRIS, via Fiorentina 1, Siena, 53100, Italy.
Lanzavecchia
S
S
Lupetti
P
P
de Bernard
M
M
Pagliaccia
C
C
Pelicic
V
V
Charrel
M
M
Ulivieri
C
C
Norais
N
N
Ji
X
X
Cabiaux
V
V
Papini
E
E
Rappuoli
R
R
Telford
J L
JL
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity / Q000648:ultrastructure*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D002470:Cell Survival; D003603:Cytotoxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D019281:Dimerization; D004705:Endocytosis; D004926:Escherichia coli / Q000235:genetics; D005613:Freeze Etching; D006367:HeLa Cells; D016480:Helicobacter pylori; D006801:Humans; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity / Q000648:ultrastructure*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility; D014617:Vacuoles / Q000648:ultrastructure
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C074689:VacA protein, Helicobacter pylori