‘Til death do us part: conformational change for MLKL, disengagement from RIPK3 in necroptosis.

Necroptosis is a lytic form of programmed cell death that is dysregulated in multiple human pathologies. Originating as a host defence mechanism against invading pathogens, necroptosis is triggered by various stimuli, resulting in plasma membrane lysis and release of inflammatory cellular contents.

The complex choreography by which necroptosis occurs is not fully understood, but the principal partners in this dance of death are the receptor-interacting serine/threonine protein kinase RIPK3 (which, having first performed autophosphorylation), phosphorylates the terminal effector protein; mixed lineage kinase domain-like (MLKL).

The authors previously developed synthetic high-affinity, high-specificity binding proteins, termed Monobodies. Here, Garnish et al make use of them to identify, bind and isolate conformational variants of the human MLKL pseudokinase domain. Using IonOpticks Aurora Series columns and a Bruker timsTOF Pro as well as X-ray crystallography, they characterise interacting proteins and solve the structures in complex with specific Monobodies, identifying two distinct conformers and confirming two events in the necroptosis pathway:

  1. Endogenous MLKL associates with RIPK3 under basal conditions, and
  2. MLKL phosphorylation leads to disengagement from RIPK3 following exposure to a necroptotic stimulus.

These results support the idea that a large conformational change accompanies MLKL activation and disengagement from RIPK3 and positions it as a key regulatory step. Further support is lent to the concept that MLKL behaves as a biphasic switch and, more broadly, that pseudokinases can function as dynamic modulators in signal transduction.

Read the full paper
Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis.
Nat Commun. 12, 2211, April 13, 2021. doi: https://doi.org/10.1038/s41467-021-22400-z

Sarah E. Garnish, Yanxiang Meng, Akiko Koide, Jarrod J. Sandow, Eric Denbaum, Annette V. Jacobsen, Wayland Yeung, Andre L. Samson, Christopher R. Horne, Cheree Fitzgibbon, Samuel N. Young, Phoebe P. C. Smith, Andrew I. Webb, Emma J. Petrie, Joanne M. Hildebrand, Natarajan Kannan, Peter E. Czabotar, Shohei Koide & James M. Murphy

Commentary by Jarrod Sandow, PhD.

About the author
Jarrod has a background in biotechnology and completed his PhD at the Institute of Medical and Veterinary Science in Adelaide. He is a co-inventor of IonOpticks’ core technology and is driven towards developing innovative solutions for the global proteomics research community that will enable scientists and clinicians to discover more from their samples to accelerate advances in biological and medical research.