Abstract
Lytic cell death culminates in plasma membrane rupture, which releases large intracellular molecules to augment the inflammatory response. Plasma membrane rupture is mediated by the effector membrane protein ninjurin-1 (NINJ1)1, which polymerizes and ruptures the membrane via its hydrophilic face1-4. How NINJ1 is restrained under steady-state conditions to ensure cell survival remains unknown. Here we describe the molecular underpinnings of NINJ1 inhibition. Using cryogenic electron microscopy, we determined the structure of inactive-state mouse NINJ1 bound to the newly developed nanobody Nb538. Inactive NINJ1 forms a face-to-face homodimer by adopting a three-helix conformation with unkinked transmembrane helix 1 (TM1), in contrast to the four-helix TM1-kinked active conformation2-4. Accordingly, endogenous NINJ1 from primary macrophages is a dimer under steady-state conditions. Inactive dimers sequester the membrane rupture-inducing hydrophilic face of NINJ1 and occlude the binding site for kinked TM1 from neighbouring activated NINJ1 molecules. Mutagenesis studies in cells show that destabilization of inactive face-to-face dimers leads to NINJ1-mediated cell death, whereas stabilization of face-to-face dimers inhibits NINJ1 activity. Moreover, destabilizing 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 plasma membrane rupture and cell death.