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Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain.

ABSTRACT: Helicobacter pylori VacA, a pore-forming toxin secreted by an autotransporter pathway, causes multiple alterations in human cells, contributes to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. Here, we present a 2.4-A crystal structure of the VacA p55 domain, which has an important role in mediating VacA binding to host cells. The structure is predominantly a right-handed parallel beta-helix, a feature that is characteristic of autotransporter passenger domains but unique among known bacterial protein toxins. Notable features of VacA p55 include disruptions in beta-sheet contacts that result in five beta-helix subdomains and a C-terminal domain that contains a disulfide bond. Analysis of VacA protein sequences from unrelated H. pylori strains, including m1 and m2 forms of VacA, allows us to identify structural features of the VacA surface that may be important for interactions with host receptors. Docking of the p55 structure into a 19-A cryo-EM map of a VacA dodecamer allows us to propose a model for how VacA monomers assemble into oligomeric structures capable of membrane channel formation.

SUBMITTER: Gangwer KA

PROVIDER: S-EPMC2042200 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain.

Gangwer Kelly A KA Mushrush Darren J DJ Stauff Devin L DL Spiller Ben B McClain Mark S MS Cover Timothy L TL Lacy D Borden DB

Proceedings of the National Academy of Sciences of the United States of America 20071002 41

Helicobacter pylori VacA, a pore-forming toxin secreted by an autotransporter pathway, causes multiple alterations in human cells, contributes to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. Here, we present a 2.4-A crystal structure of the VacA p55 domain, which has an important role in mediating VacA binding to host cells. The structure is predominantly a right-handed parallel beta-helix, a feature that is charac ...[more]

PMID: 17911250

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Project description:Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. VacA, a toxin secreted by H. pylori, is comprised of two domains, designated p33 and p55. Analysis of the crystal structure of the p55 domain indicated that its structure is predominantly a right-handed parallel beta-helix, which is a characteristic of autotransporter passenger domains. Substitution mutations of specific amino acids within the p33 domain abrogate VacA activity, but thus far, it has been difficult to identify small inactivating mutations within the p55 domain. Therefore, we hypothesized that large portions of the p55 domain might be non-essential for vacuolating toxin activity. To test this hypothesis, we introduced eight deletion mutations (each corresponding to a single coil within a beta-helical segment spanning VacA amino acids 433-628) into the H. pylori chromosomal vacA gene.All eight of the mutant VacA proteins were expressed by the corresponding H. pylori mutant strains and underwent proteolytic processing to yield ~85 kDa passenger domains. Three mutant proteins (VacA Delta484-504, Delta511-536, and Delta517-544) were secreted and induced vacuolation of mammalian cells, which indicated that these beta-helical coils were dispensable for vacuolating toxin activity. One mutant protein (VacA Delta433-461) exhibited reduced vacuolating toxin activity compared to wild-type VacA. Other mutant proteins, including those containing deletions near the carboxy-terminal end of the beta-helical region (amino acids Val559-Asn628), exhibited marked defects in secretion and increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded.These results indicate that within the beta-helical segment of the VacA p55 domain, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in protein misfolding and impaired secretion. We propose that non-essential beta-helical coils and a carboxy-terminal beta-helical segment required for proper protein folding and secretion are features shared by numerous autotransporter passenger domains.

Project description:Some strains of Helicobacter pylori are known to produce an extracellular cytotoxin that causes vacuolization in various mammalian cells. In this study, we found that concentrated culture supernatants from four Helicobacter strains isolated from patients infected with the bacterium, but having normal gastric mucosa, lacked cytotoxic activity. We also show that a higher percentage of strains isolated from patients with polymorphonuclear leukocyte infiltration of gastric mucosa are toxin positive (78%) versus those isolated from patients lacking such infiltration (33%). In addition to examining the relationship between pathology and cytotoxic activity, we used the previously published N-terminal sequence of the protein to clone and characterize vacA, the structural gene encoding the cytotoxin. Briefly, three oligonucleotides capable of encoding the first nine amino acids corresponding to the sense strand and four oligonucleotides corresponding to the noncoding strand of the last seven known amino acids of the cytotoxin protein were made. They were used in all 12 possible combinations in 12 different PCR reactions, with DNA from a cytotoxin-positive strain as template. In four combinations, the expected 69-bp fragment was seen. The sequence of this 69-bp fragment confirmed that it encoded the known N-terminal sequence of the cytotoxin. This gene is capable of encoding a 136-kDa protein with a 33-amino-acid signal peptide, whereas the purified cytotoxin is only 87 kDa, suggesting processing in the C-terminal region of the protein. A single copy of the vacA gene encodes the cytotoxin in H. pylori. Consequently, the insertion of a kanamycin resistance marker in the vacA gene produced an isogenic mutant lacking the cytotoxic activity. This mutant provides genetic evidence that vacA encodes the cytotoxin. Sequence analysis of the DNA adjacent to the vacA gene demonstrated that this gene is next to a putative cysteinyl tRNA synthetase gene. From the sequence arrangement, we predict that there are no other genes transcribed together with vacA. We also show that five of seven cytotoxin-negative strains examined still carry the sequences encoding it whereas the other two have suffered a deletion of the vacA gene. We further show that in at least one cytotoxin-negative but vacA-positive strain (MO19), there are variations in the length of the vacA gene that could explain the cytotoxin-negative phenotype in this strain.

Dataset Information

Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain.

Publications

Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain.

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