Assembly and topography of the prepore complex in cholesterol-dependent cytolysins

被引:88
作者
Heuck, AP
Tweten, RK
Johnson, AE
机构
[1] Texas A&M Univ Syst Hlth Sci Ctr, Coll Med, Dept Med Biochem & Genet, College Stn, TX 77843 USA
[2] Univ Oklahoma, Hlth Sci Ctr, Dept Microbiol & Immunol, Oklahoma City, OK 73190 USA
[3] Texas A&M Univ, Dept Chem, College Stn, TX 77843 USA
[4] Texas A&M Univ, Dept Biochem & Biophys, College Stn, TX 77843 USA
关键词
D O I
10.1074/jbc.M303151200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cholesterol-dependent cytolysins are a family of pore-forming proteins that have been shown to be virulence factors for a large number of pathogenic bacteria. The mechanism of pore formation for these toxins involves a complex series of events that are known to include binding, oligomerization, and insertion of a transmembrane beta-barrel. Several features of this mechanism remain poorly understood and controversial. Whereas a prepore mechanism has been proposed for perfringolysin O, a very different mechanism has been proposed for the homologous member of the family, streptolysin O. To distinguish between the two models, a novel approach that directly measures the dimension of transmembranes pores was used. Pore formation itself was examined for both cytolysins by encapsulating fluorescein-labeled peptides and proteins of different sizes into liposomes. When these liposomes were re-suspended in a solution containing anti-fluorescein antibodies, toxin-mediated pore formation was monitored directly by the quenching of fluorescein emission as the encapsulated molecules were released, and the dyes were bound by the antibodies. The analysis of pore formation determined using this approach reveals that only large pores are produced by perfringolysin O and streptolysin O during insertion (and not small pores that grow in size). These results are consistent only with the formation of a prepore complex intermediate prior to insertion of the transmembrane beta-barrel into the bilayer. Fluorescence quenching experiments also revealed that PFO in the prepore complex contacts the membrane via domain 4, and that the individual transmembrane beta-hairpins in domain 3 are not exposed to the nonpolar core of the bilayer at this intermediate stage.
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页码:31218 / 31225
页数:8
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