Department of Chemistry, University of Waterloo, 200 University Ave. W, Waterloo, ON N2L 3G1, Canada.
Biochem Cell Biol. 2012 Dec;90(6):709-17. doi: 10.1139/o2012-029. Epub 2012 Sep 27.
The bacterial toxin pyolysin (PLO) belongs to the family of cholesterol-dependent cytolysins (CDCs), which form large, ring-shaped oligomeric pores in cholesterol-containing membranes. Monomeric CDC molecules have a structure of four domains, with domains 2 and 3 packed against each other. After binding to target membranes containing cholesterol, toxin monomers oligomerize into pre-pore complexes. Trans-membrane pores form when the pre-pores insert into the lipid bilayer. Membrane insertion requires each subunit in the pre-pore to undergo a significant change in conformation, including the separation of domains 2 and 3. We here characterize a pyolysin mutant with an engineered disulfide bond between domains 2 and 3. The disulfide-tethered mutant binds to membranes but does not form oligomers. When mixed with wild type PLO, the two proteins form hybrid oligomers, which are reduced in size and arc-shaped rather than ring-shaped. With equimolar mixtures or the disulfide mutant in slight excess, the hybrid oligomers retain pore-forming activity, while a larger excess of the mutant suppresses pore formation. These results support a "partially cooperative" mode of protein activity, in which a limited number of functional subunits within an oligomer have to cooperate to initiate membrane insertion and pore formation.
细菌毒素白细胞素（PLO）属于胆固醇依赖性细胞溶素（CDCs）家族，该家族在含有胆固醇的膜中形成大的、环形的寡聚体孔。单体 CDC 分子具有四个结构域的结构，结构域 2 和 3 彼此紧密结合。在与含有胆固醇的靶膜结合后，毒素单体寡聚形成前孔复合物。当预孔插入脂质双层时，形成跨膜孔。膜插入需要预孔中的每个亚基发生显著的构象变化，包括结构域 2 和 3 的分离。我们在这里描述了一种具有工程化二硫键的白细胞素突变体，该二硫键位于结构域 2 和 3 之间。二硫键连接的突变体与膜结合但不形成寡聚体。当与野生型 PLO 混合时，两种蛋白质形成杂交寡聚体，其尺寸减小且呈弧形而不是环形。在等摩尔混合物或二硫键突变体略有过量的情况下，杂交寡聚体保持孔形成活性，而较大过量的突变体抑制孔形成。这些结果支持一种“部分协作”的蛋白质活性模式，其中寡聚体中的有限数量的功能亚基必须协作才能启动膜插入和孔形成。
