A Plug Release Mechanism for Membrane Permeation by MLKL

Abstract

SUMMARY MLKL is crucial for necroptosis, permeabilizing membranes through its N-terminal region upon phosphorylation of its kinase-like domain by RIP3. However, the mechanism underlying membrane permeabilization is unknown. The solution structure of the MLKL N-terminal region determined by NMR spectroscopy reveals a four-helix bundle with an additional helix at the top that is likely key for MLKL function, and a sixth, C-terminal helix that interacts with the top helix and with a poorly packed interface within the four-helix bundle. Fluorescence spectroscopy measurements indicate that much of the four-helix bundle inserts into membranes, but not the C-terminal helix. Moreover, we find that the four-helix bundle is sufficient to induce liposome leakage and that the C-terminal helix inhibits this activity. These results suggest that the four-helix bundle mediates membrane breakdown during necroptosis and that the sixth helix acts as a plug that prevents opening of the bundle and is released upon RIP3 phosphorylation.

Evolutionary divergence of the necroptosis effector MLKL

“…Notably, hNTD dimerization could only measurably kill U937 cells, whereas h4HB domain dimerization killed wild-type MDF, U937, HT29 and HeLa cells, suggesting that in some cell types the brace helix encodes an inhibitory function, possibly preventing oligomerization, consistent with previous findings. 13,18 Furthermore, these data imply that there are cell-specific factors that are required to enable hMLKL to translocate to membranes, oligomerize and permeabilize membranes.…”

“…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. 13,14,18 However, a number of questions remain unanswered. It is unclear why S345D mouse MLKL is a potent killer of murine fibroblasts, while the human counterpart, T357D/S358E, induced death of HT29 cells in one study, 19 yet did not induce pronounced death of human U2OS cells unless dimerized via a fused domain.…”

“…14 Similarly, it is unclear why forced dimerization of mouse MLKL 4HB domain was required for cell death in L929, CHO and HeLa cells, 12 but not in MDFs. 10 It is also unclear why recombinant human MLKL 4HB domain exhibits a preference for substrate liposomes containing cardiolipin, high concentrations of which are considered to be confined to mitochondrial inner membranes, 14,18 even though mitochondria are dispensable for necroptotic death. 20 In addition, the human MLKL 4HB domain potently and rapidly induced membrane permeabilization in liposome assays, 13,14,18 yet a substantial delay in cell death is observed following MLKL membrane translocation.…”

“…10 It is also unclear why recombinant human MLKL 4HB domain exhibits a preference for substrate liposomes containing cardiolipin, high concentrations of which are considered to be confined to mitochondrial inner membranes, 14,18 even though mitochondria are dispensable for necroptotic death. 20 In addition, the human MLKL 4HB domain potently and rapidly induced membrane permeabilization in liposome assays, 13,14,18 yet a substantial delay in cell death is observed following MLKL membrane translocation. 15 These observations led us to investigate the differential susceptibility of different cell types to MLKL-induced necroptotic death and whether reduced susceptibility could be overcome by inducible dimerization of either full-length MLKL or the 4HB domain.…”

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

“…Recent work has demonstrated that recruitment of MLKL to RIP3 and MLKL phosphorylation results in the conformational change that leads to the exposure of its four‐helix bundle domain (FHBD) and oligomerization of MLKL (Murphy et al , 2013). Binding of the FHBD to negatively charged phosphatidylinositol phosphates has been proposed to recruit MLKL to the plasma membrane allowing it to directly permeabilize the membrane by forming a pore (Dondelinger et al , 2014; Su et al , 2014; Wang et al , 2014). Alternatively, it has been also proposed that plasma membrane‐bound MLKL recruits Ca 2+ or Na + ion channels to permeabilize the membrane (Cai et al , 2014; Chen et al , 2014).…”