title: A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain.
abstract: Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: González-Rivera
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Campbell
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Stacey A
authors.initials: SA
authors.lastname: Rutherford
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Tasia M
authors.initials: TM
authors.lastname: Pyburn
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Nora J
authors.initials: NJ
authors.lastname: Foegeding
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Barke
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Spiller
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: McClain
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Melanie D
authors.initials: MD
authors.lastname: Ohi
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA borden.lacy@vanderbilt.edu timothy.l.cover@vanderbilt.edu.
authors.forename: D Borden
authors.initials: DB
authors.lastname: Lacy
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA borden.lacy@vanderbilt.edu timothy.l.cover@vanderbilt.edu.
authors.forename: Timothy L
authors.identifier: 0000-0001-8503-002X
authors.initials: TL
authors.lastname: Cover
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods; D009154:Mutation / Q000235:genetics*; D000072417:Protein Domains / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007473:Ion Channels; C074689:VacA protein, Helicobacter pylori