Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Abstract

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein . How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecularweight, membrane-localized complex and cell death. Using alaninescanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.pseudoenzyme | RIP kinase | ATP mimetic | programmed necrosis P rogrammed necrosis or "necroptosis" has emerged in the past 5 years as a cell death mechanism that complements the conventional cell death pathway, apoptosis, in multicellular organisms. In contrast to apoptosis, necroptosis does not appear to serve an important role in multicellular organism development (1-3) but participates in the defense against pathogens and is a likely culprit in destructive inflammatory conditions (4-7). Receptor Interacting Protein Kinase-3 (RIPK3) was identified as a key effector of necroptosis in 2009 (4, 5) and its substrate, the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL), in 2012 (8, 9), but the molecular events following RIPK3-mediated phosphorylation of MLKL required to induce cell death are unclear. The RIPK1/ RIPK3/MLKL necrosome has been proposed to activate PGAM5 (phosphoglycerate mutase 5) and Drp1 (Dynamin-related protein 1) to cause mitochondrial fragmentation and cell death (10), but the requirement for PGAM5, Drp1, and mitochondria for necroptosis has been questioned (1, 11-13).We described the structure of mouse MLKL revealing that MLKL contains a C-terminal pseudokinase domain and an N-terminal four-helix bundle (4HB) domain connected by a two-helix linker (the "brace" helices) (1). Based on our mutational and biochemical analyses, we proposed that the catalytically inactive pseudokinase domain functions as a molecular switch and that RIPK3-mediated phosphorylat...

Necroptosis is a key mediator of enterocytes loss in intestinal ischaemia/reperfusion injury

“…Recent advances have shown that activation of the kinase domain of receptor‐interacting protein1 (RIP1) and the assembly of RIP1/3‐containing signalling complex (termed the necrosome) mediated necroptosis contributes to the pathogenesis in preclinical models of brain 17, 18, heart 19, 20, and kidney 21, 22 I/R injury, which can be protected by using the RIP1 kinase inhibitor necrostatin (Nec)‐1. Meanwhile, the necrosome phosphorylates the mixed lineage kinase domain‐like protein (MLKL), which subsequently results in the rapid, active, and dynamic release of cell damage‐associated molecular patterns (DAMPs) following the loss of plasma membrane integrity and promotes ongoing inflammation and secondary tissue injury 23. High‐mobility group protein B1 (HMGB1) is a typical DAMP molecule because intracellular HMGB1 is present in all nucleated cells and is critical in the homoeostasis of most living cells, whereas extracellular HMGB1 can initiate and sustain the inflammatory response through ligation of pattern recognition receptors, including receptor for advanced glycation endproducts and toll‐like receptors 24.…”

Evolutionary divergence of the necroptosis effector MLKL

“…First, expression of the isolated mouse MLKL 4HB or the complete N-terminal domain (NTD), which encompasses the 4HB domain and two "brace" helices, killed mouse dermal fibroblasts (MDFs). 10 Second, cell death occurred to a similar extent when full-length mouse MLKL harbouring the S345D mutation, that mimics activation loop phosphorylation by RIPK3, was expressed in murine fibroblasts. 5,15,17 In addition, three studies have attributed a direct membrane-permeabilization function to recombinant human MLKL 4HB domain in in vitro liposome dye-release assays.…”

“…[5][6][7][8] RIPK3 phosphorylates the pseudokinase domain of MLKL, the most terminal known essential component of the pathway, 5,6 which is believed to induce a conformational change and unleash the N-terminal four-helix bundle (4HB) domain of MLKL: an executioner domain. 5,9,10 Several models have been proposed for how this 4HB domain might induce cell death, including activation of downstream effectors, such as ion channels, 11,12 direct permeabilization of membranes and/or formation of a transmembrane pore, 13,14 all of which remain the subject of debate. The consensus from these and other studies is that in order to kill, MLKL must translocate to membranes and assemble into high molecular weight signalling complexes, which are likely to be MLKL oligomers, although the stoichiometry of these MLKL oligomers remains an open question.…”

“…The consensus from these and other studies is that in order to kill, MLKL must translocate to membranes and assemble into high molecular weight signalling complexes, which are likely to be MLKL oligomers, although the stoichiometry of these MLKL oligomers remains an open question. [10][11][12][13][14] Nonetheless, phosphorylation appears to be a key cue for MLKL activation 15 and, as the most terminal known post-translational modification in the pathway, could potentially be utilized as a biomarker in pathologies arising from necroptotic cell death. 14,16 A model whereby RIPK3-mediated phosphorylation of the MLKL pseudokinase domain activation loop (S345 in mouse; T357/S358 in human) leads to unleashing of the executioner 4HB domain is supported by several lines of evidence.…”

Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways

“…41,42 Both Ripk3 − / − Casp8 − / − and Mlkl − / − Casp8 − / − MDFs were completely resistant to IFNγ/ SM-induced cell death, suggesting that IFNγ/SM treatment causes a caspase-8-dependent apoptosis ( Figure 3a). Inhibition of necroptosis using the MLKL inhibitor, compound 1, 43 and the RIPK1 inhibitor, Nec-1 (necrostatin-1), 44 had no impact on the sensitivity to IFNγ/SM killing ( Figure 3b), but provided some protection when combined with the caspase inhibitor, QVD ( Figure 3c). QVD alone did not stop IFNγ/SM killing because by preventing apoptosis it triggered necroptosis ( Figure 3c).…”