title: Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering.
abstract: Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation. Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, LE1 7RH, UK. gilbert@strubi.ox.ac.uk
authors.forename: R J
authors.initials: RJ
authors.lastname: Gilbert
authors.forename: R K
authors.initials: RK
authors.lastname: Heenan
authors.forename: P A
authors.initials: PA
authors.lastname: Timmins
authors.forename: N A
authors.initials: NA
authors.lastname: Gingles
authors.forename: T J
authors.initials: TJ
authors.lastname: Mitchell
authors.forename: A J
authors.initials: AJ
authors.lastname: Rowe
authors.forename: J
authors.initials: J
authors.lastname: Rossjohn
authors.forename: M W
authors.initials: MW
authors.lastname: Parker
authors.forename: P W
authors.initials: PW
authors.lastname: Andrew
authors.forename: O
authors.initials: O
authors.lastname: Byron
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009502:Neutrons; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation; D012996:Solutions; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000235:genetics; D013301:Streptolysins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation; D014867:Water / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003603:Cytotoxins; D008051:Lipid Bilayers; D008081:Liposomes; D012996:Solutions; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; D014867:Water