Structural analysis of the protein/lipid complexes associated with pore formation by the bacterial toxin pneumolysin
Structural analysis of the protein/lipid complexes associated with pore formation by the bacterial toxin pneumolysin
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DOI：
10.1074/jbc.m005126200
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发表时间：
2001-02-23
影响因子：
4.8
通讯作者：
Watts, A
中科院分区：
文献类型：
--
作者：
Bonev, BB;Gilbert, RJC;Watts, A
溶血素是人类病原体肺炎链球菌（Streptococcus pneumoniae）的主要毒力因子，是通过形成环状寡聚体来破坏含胆固醇的细胞膜的可溶性蛋白质。采用魔角自旋和宽线静态（31）pNMR结合冷冻断裂电镜研究肺炎球菌溶血素对含胆固醇、磷脂酰胆碱和磷酸双鲸蜡酯（10：10：1摩尔比）的完全水合模型膜的影响。NMR谱表明肺炎球菌溶血素与含胆固醇的脂质体相互作用导致非双层磷脂相的形成和囊泡聚集。非双层相的量随着蛋白质浓度的增加而增加。冷冻断裂电子显微镜表明聚集的囊泡和自由的环形结构的肺炎球菌溶血素的存在下共存。基于它们的大小和NMR谱的分析，可以得出结论，这些环是与脂质（每个具有840-1400个脂质）复合的肺炎球菌溶血素低聚物（含有30-50个单体）。磷脂在双层缔合复合物或游离肺炎球菌溶血素-脂质复合物中的寿命> 15 ms。进一步得出结论，肺炎球菌溶血素对脂质膜的作用是双层内孔形成、脂质提取成游离低聚复合物、脂质体聚集和融合以及膜的去稳定化导致小囊泡形成的复杂组合。
\Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)p NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio), NMR spectra show that the interaction of pneumolysin with cholesterol-containing Liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.