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Updated: Dec 25, 2025
Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis
Published on: August 7, 2018
Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies
Emma J Petrie1,2, Richard W Birkinshaw1,2, Akiko Koide3,4
1Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
Abstract:
The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.
Insights
Synthetic binding proteins called monobodies block necroptosis by preventing the MLKL protein from reaching cell membranes. This discovery reveals a new checkpoint in necroptosis signaling critical for cell death.
Area of Science:
- Cell biology
- Molecular biology
- Immunology
Background:
- Necroptosis is a regulated cell death pathway involved in host defense and inflammatory diseases.
- Activation of necroptosis involves phosphorylation of the Mixed Lineage Kinase Domain-Like (MLKL) pseudokinase by RIPK3.
- The exact control points within necroptosis signaling remain incompletely understood.
Purpose of the Study:
- To develop novel tools to investigate necroptosis signaling.
- To identify key regulatory steps in the necroptosis pathway.
- To elucidate the function of the MLKL N-terminal "killer" domain.
Main Methods:
- Development of high-affinity monobodies targeting the human MLKL N-terminal four-helix bundle (4HB) domain.
- Expression of monobodies as genetically encoded reagents in cells.
- Biochemical assays to assess MLKL recruitment, phosphorylation, oligomerization, and membrane translocation.
- X-ray crystallography to determine the structural basis of monobody binding.
- Mutational analysis to assess the role of the identified binding site in necroptosis.
Main Results:
- Monobodies were generated that bind human MLKL's 4HB domain with nanomolar affinity.
- Genetically encoded monobodies effectively inhibited necroptotic cell death.
- Monobodies blocked MLKL translocation to cellular membranes but did not impede its recruitment to the necrosome, phosphorylation by RIPK3, or oligomerization.
- X-ray crystallography identified a critical binding site on the MLKL α4 helix within the 4HB domain.
- Mutational analysis confirmed the α4 helix is essential for necroptosis signaling.
Conclusions:
- The α4 helix of the MLKL 4HB domain is a crucial regulatory site in necroptosis.
- This site is distinct from known phospholipid-binding sites and is implicated in recruiting adaptors for membrane translocation.
- Monobodies targeting this site represent potent inhibitors of necroptosis and valuable research tools.
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