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

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.

Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues

“…One possibility is that the R242A mutation enables constitutive activation via mouse RIPK3-mediated phosphorylation of horse MLKL, although currently this is not possible to test experimentally owing to the unavailability of suitable antibodies for phospho-horse MLKL. Another possibility is that the R242A substitution prompts a conformational change in the horse MLKL pseudokinase domain that unleashes the MLKL's killer 4HB domain, or facilitates exposure of MLKL to necroptosis coeffectors [53][54][55] , to enable cell death to ensue. In contrast to mouse MLKL, where mutations in the "pseudoactive" site can trigger constitutive cell death, the horse MLKL pseudokinase domain adopts a conventional active-like kinase domain fold similar to human MLKL.…”

“…MLKL contains an N-terminal four-helix bundle (4HB) domain, which enables MLKL's membrane translocation 21 and is responsible for the plasma membrane permeabilization that characterizes necroptotic cell death 18,[22][23][24][25] . The 4HB domain executioner function is regulated by the C-terminal pseudokinase domain 25 , which serves as a receiver for upstream signals, such as activation loop phosphorylation by RIPK3 18,19,26,27 .…”

Necroptosis in Immuno-Oncology and Cancer Immunotherapy

“…Herein, necroptosis is specifically triggered via necrosome-elicited oligomerization of MLKL [114][115][116]. The activated MLKL then migrates toward the plasma membrane, to create membrane pores (accompanied by "ballooning" of the plasma membrane) that facilitate massive calcium ions (Ca 2+ ) influx thereby causing the rupture of the plasma membrane ( Figure 2) [117][118][119][120]. Besides the plasma membrane disruption, MLKL may also target the mitochondrial membrane and induce mitochondrial disruption by opening the mitochondrial permeability transition pore (mPTP) [121,122].…”

A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

“…As several other necroptotic proteins are lysine-rich, we considered whether the choice of fixative was a critical variable for robust immunodetection of MLKL, RIPK3 and RIPK1 in human and mouse cells. Accordingly, we compared the performance of 22 antibodies for immunofluorescent staining of human HT29 cells and mouse dermal fibroblasts (MDFs) -two cellular models that are well-characterized to undergo necroptosis when treated with TNF, Smacmimetic and IDN-6556 (herein referred to as TSI) 5,28,29,44,47 . To quantitatively gauge the performance of each antibody, their immunosignals were characterized in four ways: 1) A ratio of the immunofluorescent signals between a positive and negative control.…”

“…In cellular contexts where the activities of the Inhibitors of Apoptosis proteins (IAPs) E3 Ubiquitin ligase family and the proteolytic apoptotic effector, Caspase-8, are depleted or compromised, necroptosis ensues. The precise choreography of necroptotic signaling is still emerging, although recent studies have defined key events and checkpoints in the pathway 26,27,28,29 . Following pathway induction, RIPK1 autophosphorylation prompts hetero-oligomerization with RIPK3 via an amyloid-forming motif in the region C-terminal to their kinase domains termed the RHIM (RIP Homotypic Interaction Motif) to form a cytoplasmic platform known as the necrosome 30,31,32 .…”