Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease.

具有死亡结构域的 Fas 相关蛋白 (FADD)、procaspase-8 和细胞 FLICE 抑制蛋白 (cFLIP) 通过死亡效应结构域 (DED) 组装，引导死亡受体信号传导至细胞存活或凋亡。由于三元 DED 复合物缺乏原子坐标，对它们三维调控机制的理解受到限制。通过采用 X 射线晶体学和低温电子显微镜 (cryo-EM)，我们展示了人类 FADD-procaspase-8-cFLIP 复合物的原子坐标，揭示了这些关键相互作用的结构见解。这些结构说明了 FADD 和 cFLIP 如何协调包含 caspase-8 的复合物的组装，并为它们在促进或抑制细胞凋亡和坏死性凋亡信号传导中的作用提供机制解释。复合物中的螺旋型 procaspase-8-cFLIP 异双层似乎可以促进有限的 caspase-8 激活以促进细胞存活。我们的结构引导诱变支持三重 FADD 复合物在 caspase-8 激活和调节受体相互作用蛋白激酶 1 (RIPK1) 中的作用。这些结果提出了 DED 组装和 procaspase-8 激活在调节涉及发育、先天免疫和疾病的各种细胞途径中的凋亡和坏死性信号传导中的统一机制。