Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering

Pneumolysin, an important virulence factor of the human pathogen Streptococ cus 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 th e oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer m embrane structure and the nature of the self-association into oligomers und ergone by it. We have used analytical ultracentrifugation (AUC) to investig ate oligomerization and small-angle neutron scattering (SANS) to investigat e the changes in membrane structure accompanying pore formation. Pneumolysin self-associates in solution to form oligomeric structures appar ently similar to those which appear on the membrane coincident with pore fo rmation. It has previously been demonstrated by us using site-specific chem ical derivatization of the protein that the self-interaction preceding olig omerization involves its C-terminal domain. The AUC experiments described h ere involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-termi nal domain leads to oligomerization and that this may be related to the mec hanism by which pneumolysin activates the complement system. SANS data at a variety of neutron contrasts were obtained from Liposomes used as model ce ll membranes in the absence of pneumolysin, and following the addition of t oxin at a number of concentrations. These experiments were designed to allo w visualization of the effect that pneumolysin has on bilayer membrane stru cture resulting from oligomerization into a pore-forming complex. The struc ture of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scatt ering curves. The fitting equations describe scattering from simple three-d imensional scattering volume models for the structures present in the sampl e, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, whic h was countered by the formation of localized structures thicker than the L iposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomer s were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in sifts b rought about changes in liposome structure similar to those seen in the pre sence 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/mem brane interactions in general. We discuss these results in the light of pub lished data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect. (C) 1999 Academic Press.