The structural basis for membrane binding and pore formation by lymphocyte perforin (3NSJ)

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.

自然杀伤细胞和细胞毒性T淋巴细胞承担着至关重要的功能，即消灭病毒感染细胞和肿瘤细胞。它们通过从细胞质颗粒中释放穿孔素和颗粒酶蛋白酶进入紧邻的杀伤细胞与靶细胞膜之间形成的间隙中来实现这一功能。穿孔素，一种分子量为67千道尔顿的多结构域蛋白，通过寡聚化形成孔道，将促凋亡的颗粒酶递送至靶细胞的细胞质中。穿孔素的重要性通过先天性穿孔素缺乏症的致命后果而得到凸显，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生症（2型FHL）相关。在本研究中，我们通过确定单体小鼠穿孔素的X射线晶体结构和整个穿孔素孔道的冷冻电子显微镜重建，阐明了穿孔素孔形成机制。穿孔素是一种细长的‘钥匙形’分子，由氨基末端膜攻击复合物穿孔素样（MACPF）/胆固醇依赖性细胞溶解素（CDC）结构域组成，随后是一个表皮生长因子（EGF）结构域，该结构域与极端的羧基末端序列共同形成一个中央的货架状结构。C端C2结构域介导初始的钙离子依赖性膜结合。然而，出人意料的是，冷冻电子显微镜揭示，穿孔素MACPF结构域在孔中的取向相对于CDC中的亚基排列是向外翻转的。这些数据揭示了保守的MACPF/CDC结构域在作用机制上的显著灵活性，并为相关免疫防御分子，如补体蛋白如何组装成孔道提供了新的见解。