Exogenous stimuli provoke assembly of the receptor-interacting serine/threonine protein kinase RIPK1:RIPK3 oligomeric complex termed the necrosome, which acts as a platform for recruiting and activating mixed lineage kinase domain-like protein (MLKL), the terminal effector pseudokinase in the necroptotic signaling pathway (Sun L et al. 2012; Zhao J et al. 2012; reviewed by Murphy JM 2020). Mass spectrometry analysis identified MLKL as a necrosome component associated with RIPK3 in a HeLa cell line in which RIPK3 was expressed and caspase-8 was knocked down to induce necrosis in the presence of necrosulfonamide (NSA) (Sun L et al. 2012). NSA was found to specifically block TNFα-induced necroptosis downstream of RIPK3 activation in human colon cancer HT-29 cells, FADD null human T cell leukemia Jurkat cells and other RIPK3-expressing cells (Sun L et al. 2012). Short hairpin (sh) RNA-mediated genetic screens targeting human kinases, phosphatases, genes involved in protein ubiquination also identified MLKL as a key RIPK3 downstream component of TNFα-induced necroptosis in HT-29 cells (Zhao J et al. 2012). Further, MLKL knockout mice and cells derived from MLKL-deficient mice demonstrated the indispensable role of Mlkl in necroptosis (Wu J et al. 2013; Murphy JM et al. 2013). MLKL knockout in human myeloid leukaemia U937cells was shown to abrogate necroptosis, while induced expression of wild-type human MLKL in MLKL-/- U937 cells restored sensitivity to the necroptotic stimulus (Petrie EJ et al. 2018; Davies KA et al. 2020). Knockdown of MLKL by shRNA in HT-29 or gastric cancer MKN45 cells inhibited tumor necrosis factor alpha (TNFα)-induced necroptosis (Sun L et al. 2012; Zhao J et al. 2012; Wang H et al. 2014). The RIPK3 kinase activity is required for interaction and activation of MLKL in necroptosis as kinase-dead RIPK3 mutants were unable to bind MLKL or mediate TNF-induced necroptosis (Zhao J et al. 2012; Murphy JM et al. 2013; Chen W et al. 2013). The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020). The pseudokinase domain (psKD) of MLKL is known to engage the kinase domain (KD) of RIPK3, stably in the case of the human system (Sun L et al. 2012; Zhao J et al. 2012; Davies KA et al. 2018; Petrie EJ et al. 2018), but transiently in the mouse system (Murphy JM et al. 2013; Chen W et al. 2013; Petrie EJ et al. 2019a). Structural studies of the mouse MLKL pseudokinase domain in complex with the mouse RIPK3 kinase domain revealed juxtaposition of RIPK3 active site next to the pseudoactive site of mouse MLKL for phosphorylation of the latter’s activation loop (Xie T et al. 2013). The KD:psKD complex is governed by extensive lobe-to-lobe interaction interfaces, stabilized by hydrophobic and electrostatic interactions. The C-lobe interface is mediated by mouse RIPK3 autophosphorylated residues. It was observed that F373 of mouse MLKL projects from the αF-αG loop into a cavity adjacent to αG in RIPK3 (Xie T et al. 2013). The structure of the human RIPK3:MLKL complex has not been determined, but its modeling based on the mouse complex suggests that similar interaction may occur, governed by different electrostatic surface potentials (Petrie EJ et al. 2019b). Ala substitution of the equivalent human MLKL residue, F386, abrogated reconstitution of necroptotic signaling in MLKL-/- U937 cells, suggesting more broadly that this C-lobe:C-lobe interaction underpins RIPK3 engagement by MLKL (Petrie EJ et al. 2019b).
MLKL is composed of an amino-terminal four-helix bundle (4HB) domain, a two-helix “brace” region, and a carboxy-terminal pseudokinase domain (Murphy JM et al. 2013; Petrie EJ et al. 2018). The 4HB domain functions as the executioner domain by virtue of its membrane permeabilization activity (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Hildebrand JM et al. 2014; Su L et al. 2014; Wang H et al. 2014; Tanzer MC et al. 2016). The 4HB domain enables membrane translocation of MLKL and is responsible for the plasma membrane permeabilization that characterizes necroptotic cell death (Chen X et al. 2014; Cai Z et al. 2014; Dondelinger Y. et al. 2014; Hildebrand JM et al. 2014; Petrie EJ et al. 2020). The 4HB domain executioner function is regulated by the C-terminal pseudokinase domain, which serves as a receiver for upstream signals, such as activation loop phosphorylation by RIPK3 (Hildebrand JM et al. 2014; Sun L et al. 2012; Rodriguez DA et al. 2016; Petrie EJ et al. 2018). Studies using mouse:human MLKL chimeras showed that the first brace helix and the adjacent loop (that connect the 4HB to the pseudokinase domain) of MLKL mediate interdomain communication and oligomerisation upon RIPK3-mediated activation of MLKL (Davies KA et al. 2018). RIPK3-mediated phosphorylation is thought to trigger a conformational change within the pseudokinase of MLKL that promotes 4HB domain exposure, enabling MLKL to form oligomers, which are trafficked to the plasma membrane where cell permeabilization occurs (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2020; Samson AL et al. 2020). Even though the Reactome annotation shows that 4 molecules of MLKL bind to the RIPK1:RIPK3 oligomer, the exact stoichiometry of the binding and the oligomerization of MLKL has been highly debated (Chen X et al. 2014; Cai Z et al. 2014; Davies KA et al. 2018; Petrie EJ et al. 2018; Petrie EJ 2017). While trimers, tetramers, hexamers were reported in studies with the recombinant MLKL protein, single-cell imaging approaches revealed that endogenous human phosphorylated MLKL assembles into higher order species that are heterogeneous in MLKL stoichiometry (Samson AL et al. 2020). The mechanisms of necroptosis regulation and execution downstream of MLKL remain elusive.