The structural basis for membrane binding and pore formation by lymphocyte perforin
The structural basis for membrane binding and pore formation by lymphocyte perforin
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DOI：
10.1038/nature09518
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发表时间：
2010-11-18
期刊：
影响因子：
64.8
通讯作者：
Whisstock, James C.
中科院分区：
文献类型：
--
作者：
Law, Ruby H. P.;Lukoyanova, Natalya;Whisstock, James C.
自然杀伤细胞和细胞毒性 T 淋巴细胞完成杀死病毒感染细胞和肿瘤细胞的至关重要的功能。他们通过将成孔蛋白穿孔素和颗粒酶蛋白酶从细胞质颗粒释放到邻接的杀伤细胞膜和靶细胞膜之间形成的裂缝中来实现这一点。穿孔素是一种 67 千道尔顿的多结构域蛋白，寡聚形成孔，将促凋亡颗粒酶递送到靶细胞的胞质溶胶中(1-6)。先天性穿孔素缺乏症的致命后果凸显了穿孔素的重要性，超过 50 种不同的穿孔素突变与家族性噬血细胞性淋巴组织细胞增多症（2 型 FHL）相关(7)。在这里，我们通过确定单体鼠穿孔素的 X 射线晶体结构以及整个穿孔素孔的冷冻电子显微镜重建来阐明穿孔素孔形成的机制。穿孔素是一种薄薄的“钥匙形”分子，包含氨基末端膜攻击复合物穿孔素样 (MACPF)/胆固醇依赖性溶细胞素 (CDC) 结构域 (8,9)，随后是表皮生长因子 (EGF) 结构域，该结构域与末端羧基末端序列一起形成中央架状结构。 C 末端 C2 结构域介导初始的 Ca2+ 依赖性膜结合。然而，最出乎意料的是，电子显微镜显示穿孔素 MACPF 结构域在孔中的方向相对于 CDCs10,11 中的亚基排列是由内而外的。这些数据揭示了保守的 MACPF/CDC 折叠的作用机制的显着灵活性，并为补体蛋白等相关免疫防御分子如何组装到孔中提供了新的见解。
Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell(1-6). The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL)(7). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain(8,9) followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca2+-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs10,11. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.