Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Feb 18;61(4):589-601.
doi: 10.1016/j.molcel.2016.01.011. Epub 2016 Feb 4.

Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis

Giovanni Quarato  1 , Cliff S Guy  1 , Christy R Grace  2 , Fabien Llambi  1 , Amanda Nourse  3 , Diego A Rodriguez  1 , Randall Wakefield  4 , Sharon Frase  4 , Tudor Moldoveanu  5 , Douglas R Green  6
Affiliations

Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis

Giovanni Quarato et al. Mol Cell. .

Abstract

Necroptosis is a cell death pathway regulated by the receptor interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) pseudokinase. How MLKL executes plasma membrane rupture upon phosphorylation by RIPK3 remains controversial. Here, we characterize the hierarchical transduction of structural changes in MLKL that culminate in necroptosis. The MLKL brace, proximal to the N-terminal helix bundle (NB), is involved in oligomerization to facilitate plasma membrane targeting through the low-affinity binding of NB to phosphorylated inositol polar head groups of phosphatidylinositol phosphate (PIP) phospholipids. At the membrane, the NB undergoes a "rolling over" mechanism to expose additional higher-affinity PIP-binding sites responsible for robust association to the membrane and displacement of the brace from the NB. PI(4,5)P2 is the preferred PIP-binding partner. We investigate the specific association of MLKL with PIPs and subsequent structural changes during necroptosis.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Oligomerization of NB Is Essential for Necroptosis
A. NBB182 and NBB140 purified proteins were >90% pure by Coomassie-staining. B. Analytical ultracentrifugation analysis of apo NBB140 and NBB182, and NSA-treated NBB140 and NBB182 identified their oligomeric profiles. M: monomer, D: dimer, T: trimer or tetramer. The experimentally derived MW is reported for each condition as in Table S1. C. Western blot analysis of Cerulean-MLKL141-182 (Cerulean-brace) oligomerization in the presence of DSS (disuccinimidyl suberate) crosslinker after 16 h of transient transfection in Hek-293 cell line. D. Kinetics and flow cytometry analyses of necroptosis induced by the 2xFV-Flag constructs during and after 12 h of protein expression. Error bars represent the SD from the mean of triplicate samples. Data are representative of n=3 independent experiments. E. Western blot analysis of spontaneous oligomerization of NBB182 after 4 h of protein expression using a BMH (bismaleimidohexane) crosslinker. *P<0.05, **P<0.01, ****P<0.0001, (Ordinary one-way ANOVA with Tukey multi-comparison post-test). See also Figure S1 and Table S1.
Figure 2
Figure 2. NB Oligomerization Drives Plasma Membrane Localization Before Rupture
A. Fast kinetics of necroptosis in mlkl−/− MEFs induced by oligomerization of NBB140-2xFV-Venus after 12 h of Dox pretreatment. Error bars represent the SD from the mean of triplicate samples. Data are representative of n=3 independent experiments. B. Corresponding flow cytometry analysis after 120 min. Error bars represent the SD from the mean of triplicate samples. Data are representative of n=2 independent experiments. C. Live confocal microscopy revealed rapid translocation of NBB140-2xFV-Venus from the cytosol to the plasma membrane upon Dim-induced oligomerization in mlkl−/− MEFs pretreated with Dox for 12 h. Yellow: NBB140-2xFV-Venus, red: LCK-C-RFP. Bar = 10 μm. Data show one representative of n=3 independent experiments. D. Total internal reflection fluorescence of mlkl−/− MEFs expressing NBB140-2xFV-Venus and NBB140(9A)-2xFV-Venus upon 12 h Dox incubation and Dim-mediated oligomerization. Bar = 10 μm. Data show one representative of n=3 independent experiments per condition. E. Magnified view of the 4 min panel shown in D. The red arrows show protein aggregations on the cell surface. F. Summary of the hallmarks of necroptosis as seen by electron microscopy after Dim-induced oligomerization of mlkl−/− MEFs and 12 h of Dox-enforced expression of NBB140-2xFV-Venus. G. Representative 3D-SEM reconstruction of mlkl−/− MEFs expressing NBB140-2xFV-Venus after a 12-h Dox treatment at 0 and 10 min after Dim treatment. See also Figure S2 and Movies S1–S5.
Figure 3
Figure 3. NB Binds to Isolated Polar Head Groups of PIPs
A. Overlaid [15N-1H] TROSY spectra of 200 μM 15N-NBB156 ± 5 mM IP3. B. CSP of NBB156 induced by 5 mM IP3 as a function of residue number. C. Mapping of IP3-binding residues onto the NMR structure of NBB2-152 (PDB ID 2MSV). The thickness of the tubes represent the magnitude of the CSP in panel B. D. Flow cytometry analysis of necroptosis induced after 12 h by Dox-enforced expression of WT and IP3-binding mutant NBB182-GFP constructs. Data are the average and SD of n=3 independent experiments in triplicate. E. Kinetics of necroptosis induced by 12 h of Dox-enforced expression of the constructs in panel D. Error bars represent the SD from the mean of triplicate samples. *P<0.05, ****P<0.0001, (Ordinary one-way ANOVA with Tukey multi-comparison post-test). See also Figure S3.
Figure 4
Figure 4. IP3-Binding Sites Recruit MLKL to Plasma Membrane
A. Western blot analysis after 12 h of Dox-enforced expression of WT and 5A mutant NBB140-2xFV-Venus constructs in mlkl−/− MEFs. B. Fast kinetics of necroptosis induced at 120 min of Dim-induced oligomerization of WT and 5A mutant NBB140-2xFV-Venus constructs after 12 h of Dox-enforced expression in mlkl−/− MEFs. Error bars represent the SD from the mean of triplicate samples. Data are representative of n=3 independent experiments. C. Corresponding flow cytometry analysis after 120 min. Data are the average and SD of n=3 independent experiments in triplicate. D. Live microscopy of early time points of necroptosis induced by Dim-mediated oligomerization in mlkl−/− MEFs after 12 h of Dox-enforced expression of WT and 5A mutant NBB140-2xFV-Venus. Yellow: Venus, Red: LCK-C-RFP. Bar = 20 μm. Data show one representative of n=3 independent experiments per condition.****P<0.0001, (Ordinary one-way ANOVA with Tukey multi-comparison post-test). See also Figure S4.
Figure 5
Figure 5. Plasma Membrane Targeting Bypasses Requirement for Weak-Affinity Recruitment
A. Schematic representation of the co-expressed chimeric RIPK3 and MLKL proteins. B. Flow cytometry analysis of necroptosis after 5 h of Dox-enforced expression of Cer-2xFV-hRIPK3 in ripk3−/−mlkl−/− MEFs that express the MLKL-Venus constructs. Error bars represent the SD from the mean of triplicate samples. Data are representative of n=3 independent experiments. C. Kinetics of necroptosis after Dox-enforced expression of RIPK3 in ripk3−/−mlkl−/− MEFs expressing the WT and 5A MLKL constructs from panel A. Error bars represent the SD from the mean of triplicate samples. Data are representative of n=3 independent experiments. D. Flow cytometry analysis of necroptosis for the cells in panel C. E. Western blot analysis of WT and 5A mutant MLKL-Venus constructs in ripk3−/− mlkl−/− MEFs upon activation by RIPK3. Data are the average and SD of n=3 independent experiments in triplicate. *P<0.05, ****P<0.0001, (Ordinary one-way ANOVA with Tukey multi-comparison post-test). See also Figure S5.
Figure 6
Figure 6. PI(4,5)P2 Binding Displaces the Brace from NB
A. Detergent micelles were mixed with PIPs followed by NBB156 to generate micellar NBB156. B. Overlaid [15N-1H] TROSY spectra of 15N-NBB156 in micellar DDM containing PIP as indicated. The free and micelle-associated NBB156 resonances used to normalize intensities between the different concentration of detergent and PIPs are labeled and circled grey and red, respectively. C. PIP-dependent induction of intrinsic disorder in the brace was quantified for the micelle-associated resonances circled red in panel B. The plot shows average and standard deviation of three normalized intensities. Details of intensity normalization are provided in Figure S6 and Experimental Procedures. D. The unstructured region corresponding to the C terminus of NBB156 was mapped onto NBB2-152 (pink PDB ID 2MSV). Basic and acidic residues involved in the interaction with IP3 and the brace are represented as sticks. E. Surface representation of NBB2-154 highlighting the acidic nature of the brace (left and center), which masks the putative basic PI(4,5)P2-binding site (right). See also Figure S6.
Figure 7
Figure 7. Sequential PI(4,5)P2 Binding Through a “Rolling Over” Mechanism Induces Brace Displacement
A. Overlaid [15N-1H] TROSY spectra of Glu136Arg 15N-NBB156 in micellar DDM containing PIP as indicated. The free and micelle-associated NBB156 resonances used to normalize intensities between the different PIP concentrations are labeled and circled grey and red, respectively. B. PIP-dependent induction of intrinsic disorder in the brace was quantified for the micelle-associated resonances circled red in panel A. C. Schematic representation of the co-expressed RIPK3 and MLKL chimeric proteins. D. The mutated basic residue are highlighted on the cartoon representation of NBB2-154. E. Kinetics of necroptosis induced by Dim-mediated oligomerization of Cer-2xFV-RIPK3 after 5 h of Dox-enforced expression of WT and mutant MLKL-Venus constructs in ripk3−/− mlkl−/− MEFs. Error bars represent the SD from the mean of triplicate samples. Data are representative of n=3 independent experiments. F. Our revised mechanism of MLKL-mediated necroptosis involves RIPK3-mediated phosphorylation of MLKL inducing a poorly understood transition from the cytosolic monomeric conformation, elucidated by the crystal structure of FL mMLKL (PDB ID 4BTF), to a transient brace-mediated oligomeric conformation. The brace-mediated oligomerization may be the site of action for the MLKL inhibitor NSA. Brace-mediated oligomerization facilitates recruitment to the plasma membrane via low-affinity binding of Lys16 Arg17 of helix α1 to the polar head group of PI(4,5)P2. We propose that after the initial plasma membrane recruitment, there is a “rolling over” mechanism involving more extensive interactions of basic residues in helix α2 with PI(4,5)P2, resulting in displacement of the brace and its intrinsic disordering. Our study does not address steps downstream of PI(4,5)P2 binding, but others (Dondelinger et al., 2014; Su et al., 2014; Wang et al., 2014) have demonstrated that MLKL can permeabilize synthetic liposomes, supporting its role as the effector of plasma membrane permeabilization. See also Figure S7.
See this image and copyright information in PMC

References

    1. Axelrod D. Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol. 1981;89:141–145. - PMC - PubMed
    1. Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, Ward Y, Wu LG, Liu ZG. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol. 2014;16:55–65. - PMC - PubMed
    1. Chen W, Zhou Z, Li L, Zhong CQ, Zheng X, Wu X, Zhang Y, Ma H, Huang D, Li W, et al. Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem. 2013;288:16247–16261. - PMC - PubMed
    1. Chen X, Li W, Ren J, Huang D, He WT, Song Y, Yang C, Li W, Zheng X, Chen P, et al. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res. 2014;24:105–121. - PMC - PubMed
    1. Czabotar PE, Murphy JM. A tale of two domains - a structural perspective of the pseudokinase, MLKL. FEBS J. 2015 doi: 10.1111/febs.13504. - DOI - PubMed

Publication types

MeSH terms

Substances