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, NK细胞）与细胞毒性T淋巴细胞（cytotoxic T lymphocytes, CTL）承担着杀伤病毒感染细胞与肿瘤细胞这一至关重要的功能。它们通过将成孔蛋白穿孔素（perforin）与颗粒酶蛋白酶（granzyme proteases）从细胞质颗粒释放至毗邻的杀伤细胞与靶细胞膜之间形成的裂隙中来完成这一功能。穿孔素是一种分子量为67千道尔顿的多结构域蛋白，可寡聚化形成孔道，将促凋亡颗粒酶转运至靶细胞的细胞质中。先天性穿孔素缺乏会导致致命后果，目前已有超过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（type 2 FHL, 2型FHL）相关，这凸显了穿孔素的重要性。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, cryo-EM）重构模型，阐明了穿孔素孔道形成的机制。穿孔素呈纤细的“钥匙状”结构，由氨基端的膜攻击复合物穿孔素样（membrane attack complex perforin-like, MACPF）/胆固醇依赖溶细胞素（cholesterol dependent cytolysin, CDC）结构域，以及随后的表皮生长因子（epidermal growth factor, EGF）结构域组成；该EGF结构域与最末端的羧基端序列共同形成中央的架状结构。羧基端的C2结构域介导穿孔素初始的钙离子依赖型膜结合过程。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结构域的取向与胆固醇依赖溶细胞素（CDC）中的亚基排列方向呈内翻状态。本研究数据揭示了保守的MACPF/CDC折叠结构域的作用机制具有显著的灵活性，并为补体蛋白等相关免疫防御分子如何组装成孔道提供了新的见解。