Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies
- 31 March 2020
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 117 (15), 8468-8475
- https://doi.org/10.1073/pnas.1919960117
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.Funding Information
- Department of Health | National Health and Medical Research Council (1172929)
- Department of Health | National Health and Medical Research Council (541951)
- Department of Health | National Health and Medical Research Council (1079700)
- Department of Health | National Health and Medical Research Council (1105754)
- Department of Health | National Health and Medical Research Council (1124735)
- Department of Health | National Health and Medical Research Council (1124737)
- Department of Health | National Health and Medical Research Council (9000433)
This publication has 59 references indexed in Scilit:
- How good are my data and what is the resolution?Acta crystallographica. Section D, Structural biology, 2013
- Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosisProceedings of the National Academy of Sciences of the United States of America, 2012
- Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 KinaseCell, 2012
- Teaching an Old Scaffold New Tricks: Monobodies Constructed Using Alternative Surfaces of the FN3 ScaffoldJournal of Molecular Biology, 2012
- Joint X-ray and neutron refinement withphenix.refineActa crystallographica. Section D, Structural biology, 2010
- Features and development of CootActa crystallographica. Section D, Structural biology, 2010
- Integration, scaling, space-group assignment and post-refinementActa crystallographica. Section D, Structural biology, 2010
- Phosphorylation-Driven Assembly of the RIP1-RIP3 Complex Regulates Programmed Necrosis and Virus-Induced InflammationCell, 2009
- Inference of Macromolecular Assemblies from Crystalline StateJournal of Molecular Biology, 2007
- Phasercrystallographic softwareJournal of Applied Crystallography, 2007