Autoinhibition of dimeric NINJ1 prevents plasma membrane rupture

• Published in: Nature (London)
• Main Authors: Pourmal, Sergei, Truong, Melissa E, Johnson, Matthew C, Yang, Ying, Zhou, Lijuan, Alegre, Kamela, Stowe, Irma B, Gupta, Shalini, Chen, Phoebe A, Zhang, Yingnan, Rohou, Alexis, Newton, Kim, Kayagaki, Nobuhiko, Dixit, Vishva M, Deshpande, Ishan
• Format: Journal Article
• Language: English
• Published: England 30-10-2024
• ISSN: 0028-0836; 1476-4687; 1476-4687
• DOI: 10.1038/s41586-024-08273-4

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)
, which polymerises and ruptures the membrane via its hydrophilic face
. 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
. 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.