Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Abstract

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.

Keywords: ATP mimetic; RIP kinase; programmed necrosis; pseudoenzyme.

Fig. 1.

The 4HB domain of MLKL is sufficient to induce necroptosis. (A) Schematic representing the different constructs used and the corresponding MLKL structure. (B–L) MDF cell lines derived from wild-type or Mlkl^−/− mice were stably infected with the indicated MLKL constructs. MLKL variants were induced for 4 h (white bars) or not (black bars), then either left untreated (UT) or treated with the apoptotic stimulus (TS) or necroptotic stimulus (TSQ) for 24 h. Q, Q-VD-OPh; S, Smac-mimetic; T, TNF. PI-permeable cells were quantified using flow cytometry. (G) As above, but in addition, cells were also treated with N, Necrostatin-1. Data are plotted as the mean ± SEM of at least two biological replicates each assayed in a minimum of three independent experiments.

Fig. 2.

Residues required for 4HB killing cluster into two motifs. (A) Alignment of MLKL orthologs. Groups of mutated residues are indicated by lowercase letters. Residues in red when mutated to Alanine prevented 4HB killing in both Mlkl^−/− and wild-type cells; mutation of residues highlighted in orange prevented 4HB killing in Mlkl^−/− but not wild-type cells. (B) Three biologically independent MDF cell lines derived from Mlkl^−/− mice were stably infected with the indicated doxycycline-inducible 4HB MLKL wild-type and mutant constructs and each assayed in two independent experiments. Cell lines were induced for 20 h (white bars) or not (black bars) before viability was quantitated. All data are plotted as mean ± SEM. (C) Depiction of the 4HB domain (Protein Data Bank ID code 4BTF) (1) drawn using PyMOL software with residues colored according to the scheme in panel A.

Fig. 3.

MLKL and the 4HB domain of MLKL form high-molecular-weight complexes in biological membranes. (A) Blue-Native PAGE showed that endogenous MLKL translocated from cytoplasm (C) to membrane (M) fraction in wild-type MDFs following TSQ treatment. (B) MLKL(1–180) similarly translocated to the membranes of Mlkl^−/− MDFs following induction. (C) Recombinant MLKL(1–169) resolved by Blue-Native PAGE. (D) Membrane complex (complex II) formation monitored by Blue-Native PAGE after a 6-h induction of wild-type MLKL(1–180), N-FLAG–tagged MLKL(1–180), cluster 2 mutants (Y15A/E16A and C18S/C24S/C28S), and cluster 1 mutants (R63A/D65A, E102A/K103A, R105A/D105A, E109A/E110A, and LLLL112–115AAAA). Data presented were obtained from three independent experiments performed on two biological replicate cell lines.

Fig. 4.

Compound 1, a small molecule targeting the nucleotide binding site of the MLKL pseudokinase domain, retards MLKL membrane translocation and inhibits necroptosis. (A and B) Compound 1 was identified as an MLKL interactor using a thermal stability shift assay. (C) Compound 1 binding to the MLKL pseudokinase domain was validated by SPR. Sensorgrams show the kinetics of compound 1 binding (at a given concentration from 6.25 to 200 µM) to MLKL (colored curves) with fit to model overlaid (black curves). x axis, time (s); y axis, response unit (RU) levels. (D) Compound 1 and Nec-1 inhibited necroptotic death of wild-type MDFs stimulated with TSQ (1 ng/mL TNF, 500 nM compound A, 10 μM Q-VD-OPh) in a dose-dependent manner. Data shown are the mean ± SEM for three independent experiments. (E) Compound 1 (1 μM) retarded MLKL translocation to the membrane. Cytoplasmic and membrane fraction purity and protein abundance are illustrated by control blots for GAPDH and VDAC1. Data are representative of three independent repeats. (F) A model for MLKL activation and the mechanism of action of compound 1.