Abstract
Necroptosis is a type of programmed cell death executed through the plasma membrane disruption by mixed lineage kinase domain-like protein (MLKL). Previous studies have revealed that an N-terminal four-helix bundle domain (NBD) of MLKL is the executioner domain for the membrane permeabilization, which is auto-inhibited by the first brace helix (H6). After necroptosis initiation, this inhibitory brace helix detaches and the NBD can integrate into the membrane, and hence leads to necroptotic cell death. However, how the NBD is released and induces membrane rupture is poorly understood. Here, we reconstituted MLKL2–154 into membrane mimetic bicelles and observed the structure disruption and membrane release of the first brace helix that is regulated by negatively charged phospholipids in a dose-dependent manner. Using molecular dynamics simulation we found that the brace region in an isolated, auto-inhibited MLKL2–154 becomes intrinsically disordered in solution after 7 ns dynamic motion. Further investigations demonstrated that a cluster of arginines in the C-terminus of MLKL2–154 is important for the molecular conformational switch. Functional mutagenesis showed that mutating these arginines to glutamates hindered the membrane disruption of full-length MLKL and thus inhibited the necroptotic cell death. These findings suggest that the brace helix also plays an active role in MLKL regulation, rather than an auto-inhibitory domain.
Highlights
Necroptosis is a type of programmed necrosis that has been implicated in host immune defense and various human diseases [1,2]
The obstacles to observe MLKL2–154 in these two membrane systems are due to nanodiscs that are restricted by the scaffolding protein to make enough room for the MLKL2–154 to insert into the bilayers, while the size of liposomes are too large for NMR studies
Our study showed a new role of the auto-inhibitory region that is not passive in the membrane disruption, which provides a better understanding of the activation steps on Mixed lineage kinase domain-like protein (MLKL) recruited to the plasma membrane
Summary
Necroptosis is a type of programmed necrosis that has been implicated in host immune defense and various human diseases [1,2]. Mixed lineage kinase domain-like protein (MLKL) is the terminal key mediator in the necroptotic pathway discovered by Sun et al in 2012 [3]. MLKL is found in the cytosol as a monomeric pseudokinase [4]. MLKL is phosphorylated by Receptor Interacting Protein Kinase 3 (RIPK3) [5], which drives MLKL oligomerization and translocation from the cytosol to the plasma membrane and leads to cell membrane disruption. A great deal of extensive studies on MLKL have been conducted [6,7]. The precise mechanism of MLKL activation, including the oligomerization, membrane translocation and permeabilization, is still under debate
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