In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis^1,2,3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers^4,5,6,7,8,9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active ‘slinky’-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning β-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

为了应对病原体感染，gasdermin (GSDM) 蛋白形成膜孔，诱导称为焦亡的宿主细胞死亡过程^1,2,3。对人和小鼠 GSDM 孔的研究揭示了包含 24 至 33 个原聚体^4,5,6,7,8,9的组件的功能和结构，但膜靶向和 GSDM 孔形成的机制和进化起源仍然未知。在这里，我们确定了细菌 GSDM (bGSDM) 孔的结构，并定义了孔组装的保守机制。我们设计了一组用于位点特异性蛋白水解激活的 bGSDM，证明不同的 bGSDM 形成不同的孔径，范围从较小的哺乳动物样组件到包含超过 50 个原聚体的异常大的孔。我们确定了活性“紧身”状寡聚构象中Vitiosangium bGSDM的冷冻电子显微镜结构，并分析了天然脂质环境中的 bGSDM 孔，以创建完整 52 聚体 bGSDM 孔的原子级模型。将我们的结构分析与分子动力学模拟和细胞测定相结合，我们的结果支持 GSDM 孔组装的逐步模型，并表明共价结合的棕榈酰基可以在形成跨膜 β 链区域之前离开疏水鞘并插入膜中。这些结果揭示了自然界中发现的 GSDM 孔的多样性，并解释了古老的翻译后修饰在实现宿主细胞程序性死亡中的功能。