Structural analysis of heteroheptamer transmembrane pore supramolecule of staphylococcal gamma-hemolysin

葡萄球菌γ-溶血素（Staphylococcal γ-hemolysin，Hlg）是由金黄色葡萄球菌分泌的一种双组分溶细胞素。我们先前已经表明，双组分γ-溶血素由LukF（或Hlg 1，34 kDa）和Hlg 2（32 kDa）组装而成，在人红细胞上形成约200 kDa的环形跨膜孔。我们最近的研究，涉及电子显微镜和化学交联表明，LukF和Hlg 2组装在一个随机的方式，形成交替复合物的亚基化学计量为3：4和4：3。在本研究项目中，我们尝试构建纳米金标记的LukF和Hlg 2的谷胱甘肽-S-转移酶（GST）融合蛋白，以在电子显微镜下观察LukF和Hlg 2的交替排列。对于GST融合蛋白的纳米金标记，通过使用聚合酶链反应的引物延伸程序，成功地将GST的三个Cys残基（Cys 138除外）替换为Ala。由此获得的LukF和Hlg 2的重组GST融合蛋白在其对应物存在下具有溶血活性。我们现在尝试将纳米金标记的GST融合蛋白与未标记的融合蛋白和纳米金颗粒分离。在本研究计划中，我们还利用电子显微镜分析了Hlg杂七聚体的精细结构。从人红细胞中分离的孔复合物用磷钨酸负染，并在透射电子显微镜下观察。从不同角度收集和分析孔复合物的高分辨率图像。其结果是，孔复合体的Hlg具有10 nm的宽度和10 nm的高度的桶形态。

Staphylococcal γ-hemolysin (Hlg) is a two-component cytolysin secreated by Staphylococcus aureus. We have previously shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of approximately 200 kDa on human erythrocytes. Our recent study involving electron microscopy and chemical cross-linking suggested that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. In this research project, we attempted to construct nanogold-labeled glutathione-S-transferase (GST) fusion proteins of LukF and Hlg2 to visualize the alternate arrangements of LukF and Hlg2 under electron microscope. For nanogold labeling of the GST fusion proteins, three Cys residues (except for Cys138) of GST were successfully replaced with Ala by using primer extension procedure of polymerase chain reactions. Recombinant GST fusion proteins of LukF and Hlg2 thus obtained were hemolytically active in the presence of their counterpart. We now attempt to separate nanogold-labeled GST fusion proteins from unlabeled fusion proteins and nanogold particles. In this research project, we also analyzed fine structure of the heteroheptamers of Hlg by electron microscopy. The pore complexes isolated from human erythrocytes were negatively stained with phosphotungstic acid and observed under a transmission electron microscopy. High resolution images of the pore complexes from different angles were collected and analyzed. As a result, the pore complex of Hlg has a barrel morphology with 10-nm width and 10-nm height.