Lytic cell death culminates in plasma membrane rupture (PMR), which releases large intracellular molecules to augment the inflammatory response. PMR is mediated by the effector membrane protein ninjurin-1 (NINJ1)¹, which polymerises and ruptures the membrane via its hydrophilic face^1–4. How NINJ1 is restrained under steady-state conditions to ensure cell survival remains a mystery. Here we describe the molecular underpinnings of NINJ1 inhibition. Using cryogenic electron microscopy, we determined the structure of inactive-state mouse NINJ1 bound to a newly-developed nanobody, Nb538. Inactive NINJ1 forms a face-to-face homodimer by adopting a 3-helix conformation with unkinked transmembrane helix 1 (TM1), in contrast to the 4-helix TM1-kinked active conformation^2–4. Accordingly, endogenous NINJ1 from primary macrophages is a dimer under steady-state conditions. Inactive dimers sequester the PMR-inducing hydrophilic face of NINJ1 and occlude the binding site for kinked TM1 from neighbouring activated NINJ1 molecules. Mutagenesis studies in cells show that destabilisation of inactive face-to-face dimers leads to NINJ1-mediated cell death, whereas stabilisation of face-to-face dimers inhibits NINJ1 activity. Moreover, destabilising mutations prompt spontaneous TM1 kink formation, a hallmark of NINJ1 activation. Collectively, our data demonstrate that dimeric NINJ1 is autoinhibited in trans to prevent unprovoked PMR and cell death.

裂解性细胞死亡以质膜破裂 （PMR） 告终，质膜破裂释放细胞内大分子以增强炎症反应。PMR 由效应膜蛋白 ninjurin-1 （NINJ1）¹ 介导，该蛋白通过其亲水面^1-4 聚合并破裂膜。如何在稳态条件下抑制 NINJ1 以确保细胞存活仍然是一个谜。在这里，我们描述了 NINJ1 抑制的分子基础。使用低温电子显微镜，我们确定了与新开发的纳米抗体 Nb538 结合的非活性态小鼠 NINJ1 的结构。与未扭结的 TM1 活性构象^2-4 相反，无活性的 NINJ1 通过采用具有未扭结的跨膜螺旋 1 （TM1） 的 3 螺旋构象形成面对面的同型二聚体。因此，来自原代巨噬细胞的内源性 NINJ1 在稳态条件下是二聚体。失活的二聚体隔离了 NINJ1 的 PMR 诱导亲水面，并封闭了邻近活化的 NINJ1 分子的扭结 TM1 结合位点。细胞中的诱变研究表明，无活性的面对面二聚体的不稳定会导致 NINJ1 介导的细胞死亡，而面对面二聚体的稳定会抑制 NINJ1 活性。此外，不稳定突变会促使自发性 TM1 扭结形成，这是 NINJ1 激活的标志。总的来说，我们的数据表明，二聚体 NINJ1 在反式中是自抑制的，以防止无端的 PMR 和细胞死亡。