Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

Proc Natl Acad Sci U S A. 2020 Apr 14;117(15):8468-8475. doi: 10.1073/pnas.1919960117. Epub 2020 Mar 31.


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.

Keywords: RIPK3; cell death; programmed necrosis; protein engineering; protein interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomimetic Materials / pharmacology*
  • Cell Membrane / metabolism*
  • Crystallography, X-Ray
  • Fibronectin Type III Domain*
  • Humans
  • Necrosis*
  • Oligopeptides / pharmacology*
  • Phosphorylation
  • Protein Conformation
  • Protein Kinases / chemistry
  • Protein Kinases / metabolism*
  • Protein Multimerization
  • Protein Transport
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*


  • Oligopeptides
  • MLKL protein, human
  • Protein Kinases
  • RIPK3 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases

Associated data

  • PDB/6UX8