Abstract
Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.
Highlights
Necroptotic cell death is mediated by the most terminal known effector of the pathway, mixed lineage kinase domain-like (MLKL)
We previously observed that introduction of an E351K mutation, which was first identified in a lung carcinoma[19], into the recombinant pseudokinase domain (PsKD) led to enhanced ATP binding[14]
While wild-type hMLKL eluted as two distinct peaks from size exclusion chromatography (SEC), as expected[12], E351K eluted as a single species at a retention time equivalent to the 55 kDa wild-type hMLKL monomer (Supplementary Fig. 1a, b)
Summary
Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. How phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. HMLKL PsKD mutants, including some identified in cancers, stabilized a monomeric state, leading to deficiencies in membrane permeabilization in liposome dye release assays and in cell death induction. These data support the idea that mutations or binding of ligands, such as ATP, within the PsKD favor a monomeric form of hMLKL that exists basally in the cytoplasm prior to the cell receiving a necroptotic stimulus. The transition to the hMLKL tetramer was modeled from small-angle X-ray scattering (SAXS) data and molecular dynamics (MD), with intermolecular crosslinks as constraints, yielding a model in which the second brace helix serves an important role in higher-order assembly: an assertion further supported by hydrogen–deuterium exchange MS (HDX-MS)
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