The structural basis for membrane binding and pore formation by lymphocyte perforin

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. 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). 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 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, cryo-electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. 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.

自然杀伤细胞（Natural killer cells）与细胞毒性T淋巴细胞（cytotoxic T lymphocytes）执行着杀伤病毒感染细胞与肿瘤细胞这一至关重要的功能。二者通过将胞质颗粒中的成孔蛋白穿孔素（perforin）与颗粒酶蛋白酶释放至毗邻的杀伤细胞与靶细胞膜之间形成的裂隙，完成该杀伤过程。穿孔素为分子量67千道尔顿的多结构域蛋白，可寡聚化形成孔道，将促凋亡颗粒酶递送至靶细胞的胞质溶胶中。先天性穿孔素缺陷可引发致命后果，目前已有超过50种穿孔素突变与家族性噬血细胞性淋巴组织细胞增生症（2型FHL）相关，这一现象凸显了穿孔素的生理重要性。本研究通过解析单体鼠源穿孔素的X射线晶体结构，以及完整穿孔素孔道的冷冻电子显微镜（cryo-electron microscopy）重构结果，阐明了穿孔素孔道形成的分子机制。穿孔素呈细长的“钥匙形”分子，由氨基端的膜攻击复合物穿孔素样（MACPF）/胆固醇依赖型细胞毒素（CDC）结构域与表皮生长因子（EGF）结构域组成；后者与极端羧基末端序列共同构成中央支架状结构。羧基末端的C2结构域介导了钙离子依赖的初始膜结合过程。然而最出乎意料的是，冷冻电子显微镜结果显示，孔道中穿孔素MACPF结构域的取向相对于CDC的亚基排布呈外翻状态。本研究揭示了保守的MACPF/CDC折叠域在作用机制上的显著灵活性，并为补体蛋白等相关免疫防御分子如何组装形成孔道提供了全新的见解。