Autoinhibition of dimeric NINJ1 prevents plasma membrane rupture

Affiliations

• Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA, USA
• Department of Structural Biology, Genentech Inc., South San Francisco, CA, USA
• Department of Discovery Chemistry, Genentech, South San Francisco, CA, USA
• Department of Biological Chemistry, Genentech, South San Francisco, CA, USA

PMID:  39476863  DOI: 10.1038/s41586-024-08273-4

Abstract

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 face1–4. 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 conformation2–4. 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.

裂解性细胞死亡以细胞膜破裂（PMR）为终点，这一过程会释放大量细胞内分子，从而增强炎症反应。PMR的发生是由效应膜蛋白Ninjurin-1（NINJ1）介导的，NINJ1通过其亲水性面进行聚合并导致膜的破裂。目前尚不清楚NINJ1在稳态条件下是如何被抑制以确保细胞存活的。我们在这项研究中探讨了NINJ1抑制的分子机制。通过低温电子显微镜，我们确定了与新开发的纳抗体Nb538结合的非活性小鼠NINJ1的结构。非活性NINJ1以三螺旋构象形成面对面同源二聚体，这与活性状态下的弯曲跨膜螺旋1（TM1）形成四螺旋的构象相对。在稳态条件下，初级巨噬细胞中的内源性NINJ1以二聚体形式存在。这些非活性二聚体能够隔离NINJ1诱导PMR的亲水性面，并阻碍来自邻近激活NINJ1分子的弯曲TM1的结合。通过细胞内的突变研究，我们发现，非活性面对面二聚体的不稳定性会导致NINJ1介导的细胞死亡，而稳定的面对面二聚体则能够抑制NINJ1的活性。此外， destabilizing mutations 促使TM1自发发生弯曲，这是NINJ1激活的一个标志。综上所述，我们的研究表明，二聚体NINJ1通过转向自我抑制，防止无缘无故的PMR和细胞死亡。

关键词：NINJ1，NINJ1中和抗体，Nb538，中和抗体，流式抗体，NINJ1流式抗体，纳米抗体，佰乐博，佰乐博生物