Supporting Information Hildebrand et al. 10.1073/pnas.1408987111 SI Materials and Methods Expression Constructs. The mouse Mixed Lineage Kinase DomainLike (mMLKL) cDNA encoding residues 1–464 was synthesized to eliminate several restriction sites by silent substitutions (DNA2.0, CA). Wild-type and mutant MLKL cDNAs were ligated into the doxycycline (dox)-inducible, puromycin-selectable vector, pF TRE3G PGK puro, as described previously (1–3), as BamHI– EcoRI fragments. The library of mouse MLKL(1–180) mutants in the pF TRE3G PGK puro vector were synthesized by DNA2.0, CA or prepared by oligonucleotide-directed PCR mutagenesis. Mutations were introduced into full-length MLKL (encoding residues 1–464) by oligonucleotide-directed PCR. All oligonucleotides were synthesized by Integrated DNA Technologies. All sequences can be obtained upon request. Sequences were verified by Sanger sequencing (Micromon DNA Sequencing Facility) or by DNA2.0. Lentiviral particles were produced by transfecting HEK293T cells seeded in 10-cm dishes with 1.2 μg of vector DNA together with two helper plasmids (0.8 μg of pVSVg and 2 μg of pCMV ΔR8.2) as described previously (4). Viral supernatants were used to infect target cells with transfected cells selected for and maintained in 5 μg/mL puromycin. Cell Lines and Cell Death Assays. Recombinant hTNF-Fc was produced in-house as described previously (5). Mouse dermal fibroblasts (MDFs) were isolated from three Mlkl−/− mice and three congenic wild-type mice and then immortalized by SV40 large T antigen to generate three biologically independent cell lines, as described (3). Immortalized MDFs were similarly prepared from three Receptor Interacting Protein Kinase-3– deficient (Ripk3−/−) mice and congenic wild-type mice. MDFs and HEK293T were maintained in DMEM supplemented with 8–10% (vol/vol) FCS and 5 μg/mL puromycin for lines stably transduced with inducible expression constructs for MLKL. Cell death assays were carried out in 24-well plates, seeding 1 × 105 cells per well. Cells attached over 4 h in the presence of 10 ng/mL dox (for Fig. 1) were then treated with assorted combinations of Necrostatin (50 μM) and QVD-OPh (10 μM) 30 min before addition of TNF (1 ng/mL in Fig. 4B; 100 ng/mL in Figs. 3A and 4E and Figs. S4 and S5D) and Smac mimetic (500 nM). After 24 h, cells were harvested and propidium iodide (PI)-positive cells (1 μg/mL) quantified using a BD FACSCalibur flow cytometer. For Fig. 2 and Fig. S2, 10 ng/mL dox was added for 20 h before cells were harvested and PI-positive cells quantified as above. Statistical Analyses. Error bars represent mean ± SD or SEM (specified in figure legends) of a specified number of independent and/or biological repeats, not technical replicates. Fractionation and Blue-Native PAGE. MDFs were seeded in six-well plates (5 × 105 per well) and allowed to attach overnight and stimulated with TSQ for wild-type MDFs or dox to induce MLKL(1–180) expression for up to 6 h, before cells were harvested and permeabilized in buffer [20 mM Hepes (pH 7.5), 100 mM KCl, 2.5 mM MgCl2, and 100 mM sucrose] containing 0.025% digitonin (BIOSYNTH) and 2 μM N-ethyl maleimide, protease, and phosphatase inhibitors. Cytosolic and crude membrane fractions were separated by centrifugation at 11,000 × g for 5 min before the crude membrane fraction was further solubilized in permeabilization buffer + 1% digitonin and clarified by centrifugation. Digitonin was added to the cytosolic fraction (final 1% wt/vol) before both fractions were resolved on a 4–16% Bis·Tris Native PAGE gel (LifeTechnologies). FolHildebrand et al. www.pnas.org/cgi/content/short/1408987111 lowing transfer of proteins to PVDF, PVDF was destained and probed for MLKL using the 3H1 antibody after soaking the PVDF membrane in 6 M Guanidine Hydrochloride, 10 mM Tris·HCl pH 7.5, and 5 mM 2-Mercaptoethanol for 2 h at room temperature. Recombinant Protein Expression and Purification. Recombinant mouse MLKL pseudokinase domain (residues 179–464) bearing a conventional N-terminal His6 tag as encoded by the pFastBac HTb vector or a modified 2xHis6 tag, MSHHHHHHGSAGSAKKKGSAGSAHHHHHHGSA, introduced into the pFastBac1 vector were expressed and purified from Sf21 insect cells according to established procedures (3, 6, 7). Briefly, these proteins were purified from Sf21 lysates by Ni2+ affinity chromatography (Roche HisTag resin). The conventional His6 tag was then cleaved by incubation with tobacco etch virus (TEV) protease for 1 h at 25 °C, before extensive dialysis and further Ni2+ chromatography to eliminate undigested protein and TEV protease followed by Superdex-200 gel filtration chromatography (GE Healthcare). Protein was eluted in 200 mM NaCl and 20 mM Hepes pH 7.5 for thermal stability shift assays or 100 mM NaCl and 20 mM Hepes pH 7.5 for NMR studies. Uncleaved 2xHis6-tagged pseudokinase Ni2+-eluate was concentrated by centrifugal ultrafiltration and subjected to Superdex-200 gel filtration chromatography (GE Healthcare) with elution in 200 mM NaCl, 20 mM Tris pH 8, 10% (vol/vol) glycerol, and 0.5 mM TCEP [tris(2-carboxyethyl)phosphine] before use in Surface Plasmon Resonance (SPR) experiments. Recombinant mRIPK3 kinase domain was expressed and purified from Sf21 cells as described previously (3, 7). Recombinant mMLKL(1–169) was prepared from Escherichia coli (BL21 Codon Plus) using an established strategy (8, 9). Briefly, a cDNA encoding mMLKL(1–169) was ligated in frame into the Kanamycin-selectable vector, pETNusH HTb (8, 9), to enable expression as a fusion protein bearing an N-terminal, TEV protease-cleavable NusA–His6 tag. Bacteria were cultured in Super Broth containing 50 μg/mL Kanamycin at 37 °C until an OD595 ∼0.6–0.8 was reached, then the temperature was lowered to 18 °C, and 20 min later expression was induced by addition of 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). Cells were cultured for a further 16 h at 18 °C; harvested by centrifugation; resuspended in 200 mM NaCl, 5 mM imidazole, 20 mM Hepes pH 7.5, and 5 mM 2-mercaptoethanol supplemented with 1 mM PMSF; lysed by sonication; and debris eliminated by centrifugation. The supernatant was clarified by syringe-driven 0.45-μM filtration and applied to a 1-mL NiMAC cartridge (Novagen) via peristaltic pump. Following washes with 7–10 column volumes of lysis buffer and lysis buffer containing 35 mM imidazole pH 7.5, NusA–His6–mMLKL(1–169) was eluted in 200 mM NaCl, 250 mM imidazole, 20 mM Hepes pH 7.5, and 5 mM 2-mercaptoethanol and incubated for 2 h at 20 °C with 0.5 mg TEV protease to cleave mMLKL(1–169) from the fusion tag. With the exception of the TEV protease cleavage step, all other purification steps were performed at 4 °C. The cleavage reaction was then dialyzed extensively against 200 mM NaCl and 20 mM Hepes pH 7.5 to eliminate imidazole before the dialysate was recovered and reapplied to a recharged NiMAC cartridge and washed with lysis buffer. The flow-through was concentrated by centrifugal ultrafiltration and applied to a Superdex-200, 24-mL gel filtration column (GE Healthcare) and eluted in 100 mM KCl and 10 mM Tris·HCl pH 8.0 for analytical ultracentifugation (AUC) studies. 1 of 8 Thermal Shift Assays to Screen for Small-Molecule Interactors. Thermal shift assays were performed as described previously (3, 6, 10) using a Corbett Real Time PCR machine after diluting proteins to 2.6 μM in 150 mM NaCl, 20 mM Tris pH 8.0, and 1 mM DTT in a total reaction volume of 25 μL. SYPRO Orange (Molecular Probes) was used to detect protein thermal unfolding via fluorescence detected at 530 nm. ATP was added at 0.2 mM and was used as a positive control for ligand binding. Small molecules from the Published Kinase Inhibitor Set (11) (kindly provided by GSK) were added at 40 μM of final concentration. A positive ΔTm value indicates that ligand binds the protein and confers protection from denaturation. The data shown are representative of three independent experiments. SPR Binding Experiments. The kinetics of compound 1 binding to the mouse MLKL pseudokinase domain were determined by SPR on a Biacore T200 instrument (GE Healthcare). Double Histagged MLKL and an unrelated negative control reference protein were immobilized on an NTA Capture chip charged with Ni2+, according to the manufacturer’s instructions. Typical immobilization levels were 2,000–3,000 Response Units (RUs). Flow cell 1 was left blank as a reference surface. Immobilization experiments were carried out at 25 °C in a running buffer containing 20 mM Hepes (pH 8.0), 200 mM NaCl, and 0.005% (vol/vol) surfactant P20. Binding experiments were carried out in Running Buffer + 2% (vol/vol) DMSO. Six compound 1 concentrations ranging from 3.125 μM to 200 μM [in Running Buffer + 2% (vol/vol) DMSO] were flowed over immobilized proteins at a flow rate of 100 μL/min, with an association phase of 30 s and dissociation phase of 90 s. Data were reduced, solvent corrected, and double referenced by Biacore T200 Evaluation Software. Data were fit globally to a two-state kinetic interaction model and the Kd determined from the (kd/ka) ratio. A 1:1 binding stoichiometry was inferred from the steady-state binding curves and the maximum observed RU levels. AUC. AUC experiments were conducted in a Beckman model XL-I instrument at 8 °C. Recombinant MLKL(1–169) at 2.2 μM, 9 μM, and 29 μM in 10 mM Tris·HCl and 100 mM KCl pH 8.0 were loaded into double sector quartz cells and mounted in a Beckman four-hole An-60 Ti rotor. Solvent density (1.0049 g/mL at 8 °C) and viscosity (1.387 cp) and an estimate of the partial specific volume (0.7328 mL/g) were computed using the amino acid composition and the program SEDNTERP (12). For sedimentation velocity (SV) experiments, 380 μL of sample and 400 μL of reference solution were centrifuged in a Beckman Coulter An50Ti rotor at a speed of 50,000 rpm, and the data were collected at a single wavelength (280 nm) in continuous mode, using a time interval of 0 s and a step size of 0.001 cm without averaging. SV data at multiple time points were fitted to a continuous sedimentation-coefficient model (13–15) using the program SEDFIT, which is available from www.analyticalultracentrifugation.com. For sedimentation equilibrium (SE) experiments, 100 μL of sample and 120 μL of reference solution were centrifuged at rotor speeds of 13,000 and 20,000 rpm. Once equilibrium was attained (∼24 h for each speed), the data were collected at a single wavelength (251 nm) in step mode, using a step size of 0.001 cm with 10 averages. SE data were fitted to a discrete species model (assuming two species) or globally fit with velocity data (collected as described above) using simulated annealing and the Marquartd–Levenberg or Simplex fitting methods with the program SEDPHAT (16), available from www. analyticalultracentrifugation.com. Mass conservation was used in fitting. For the global fit, a series of possible models were trialed, including monomer–dimer and monomer–tetramer self-associations, neither of which fitted the data as well as the monomer– Hildebrand et al. www.pnas.org/cgi/content/short/1408987111 trimer self-association model. The errors for the fitted parameters were estimated using the F statistic method, as outlined at www. analyticalultracentrifugation.com/sedphat/statistics.htm. Saturation Transfer Difference–NMR Spectroscopy. Nucleotides were dissolved in NMR buffer [20 mM Hepes, pH 7.5, 200 mM NaCl, 90% (vol/vol) D2O, and 10% (vol/vol) H2O] at a final concentration of 200 μM in each sample of STD experiments. Three different samples were prepared for each nucleotide: (i) ATP or ADP with protein buffer added as equivalent volume of protein containing samples, (ii) ATP or ADP with MLKL pseudokinase domain at a final concentration of 2 μM, and (iii) ATP or ADP with MLKL pseudokinase domain (2 μM) with compound 1 at 200 μM. NMR spectra were recorded at 283 K on a Bruker AVANCE Ultrashield 600 MHz spectrometer fitted with a Cryoprobe. 1H chemical shifts were referenced to the 1H2O signal at 4.70 ppm. Saturation of the protein resonances was achieved by a 4-s train of Gaussian pulses centered at −0.5 ppm. For the reference spectra, a similar saturation pulse was applied at a frequency centered 20,000 Hz off-resonance. A 15-ms T2-spin-lock period was used before acquisition to allow the residual protein signal to decay. NMR data were processed in TOPSPIN version 3.2 (Bruker BioSpin). In Vitro Kinase Assays. Radiometric in vitro kinase assays were performed as described previously (3, 7), but for the addition of either a DMSO control or up to 10 μM of compound 1 in 0.5% (vol/vol) final DMSO. Cold kinase assays were performed analogously, but reacted 0.5 μg mRIPK3 kinase domain with 3.5 μg mMLKL(179–464) in a 20-μL volume in the presence of 1 mM cold ATP (without 32P-γ-ATP) and 25 μM compound 1 in a final concentration of 0.125% vol/vol DMSO (or DMSO alone) for 2 h at 25 °C. Reactions were terminated by addition of reducing loading dye and boiling for 5–10 min. Mass Spectrometry Analysis of MLKL Phosphorylation. Each cold kinase assay was loaded across two lanes of a 4–20% Tris·Glycine SDS/PAGE and electrophoresed to resolve mRIPK3 kinase domain and mMLKL(179–464). Individual bands were excised, manually in-gel reduced (10 mM TCEP), alkylated (iodoacetamide), and digested with trypsin (Promega Gold). Extracted peptides were injected and separated by nano-flow reversed-phase liquid chromatography on a nano ultraperformance liquid chromatography (UPLC) system (Waters nanoAcquity) using a nanoAcquity C18 150 mm × 0.075 mm I.D. column (Waters) with a linear 60-min gradient set at a flow rate of 0.4 μL/min from 95% solvent A (0.1% Formic acid in milliQ water) to 100% solvent B [0.1% Formic acid, 80% acetonitrile (Mallinckrodt Baker), and 20% milliQ water]. The nano UPLC was coupled online to a Q-Exactive mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) set to acquire full scan (70,000 resolution) and top-10 multiply charged species selected for fragmentation using the high-energy collision disassociation (HCD) (single-charged species were ignored). Fragment ions were analyzed with the resolution set at 17,500, with the ion threshold set to 1e5 intensity. The activation time was set to 30 ms, and the normalized collision energy was stepped ±20% and set to 26. Raw files consisting of full-scan MS and highresolution MS/MS spectra were searched using the Maxquant algorithm (version 1.4). The amino acid sequences for the expressed recombinant mRIPK3 and mMLKL proteins were added to the UniRef Uni-ProtKB/Swiss-Prot database (release-2013_12), and raw files were searched against all mouse entries. Trypsin was set to two missed cleavages, and files were searched with variable modifications set for oxidized methionine, phosphorylated S/T/Y, and fixed modification in the form of carbamidomethyl Cys residues (using the default Maxquant settings with the cutoff score and delta score for modified peptides set at 40 and 17, respectively). 2 of 8 1. Moujalled DM, Cook WD, Murphy JM, Vaux DL (2014) Necroptosis induced by RIPK3 requires MLKL but not Drp1. Cell Death Dis 5:e1086. 2. Moujalled DM, et al. (2013) TNF can activate RIPK3 and cause programmed necrosis in the absence of RIPK1. Cell Death Dis 4:e465. 3. Murphy JM, et al. (2013) The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39(3):443–453. 4. Vince JE, et al. (2007) IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell 131(4):682–693. 5. Bossen C, et al. (2006) Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human. J Biol Chem 281(20):13964–13971. 6. Murphy JM, et al. (2014) A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. Biochem J 457(2):323–334. 7. Cook WD, et al. (2014) RIPK1- and RIPK3-induced cell death mode is determined by target availability. Cell Death Differ 21(10):1600–1612. 8. Hercus TR, et al. (2013) High yield production of a soluble human interleukin-3 variant from E. coli with wild-type bioactivity and improved radiolabeling properties. PLoS ONE 8(8):e74376. 9. Murphy JM, Metcalf D, Young IG, Hilton DJ (2010) A convenient method for preparation of an engineered mouse interleukin-3 analog with high solubility and wildtype bioactivity. Growth Factors 28(2):104–110. 10. Murphy JM, et al. (2014) Insights into the evolution of divergent nucleotide-binding mechanisms among pseudokinases revealed by crystal structures of human and mouse MLKL. Biochem J 457(3):369–377. 11. Drewry DH, Willson TM, Zuercher WJ (2014) Seeding collaborations to advance kinase science with the GSK Published Kinase Inhibitor Set (PKIS). Curr Top Med Chem 14(3): 340–342. 12. Laue TM, Shah BD, Ridgeway TM, Pelletier SL (1992) Computer-Aided Interpretation of Analytical Ultracentrifugation Data for Proteins. Analytical Ultracentrifugation in Biochemistry and Polymer Science (Royal Society of Chemistry, Cambridge, UK), pp 90–125. 13. Perugini MA, Schuck P, Howlett GJ (2000) Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid. J Biol Chem 275(47): 36758–36765. 14. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78(3):1606–1619. 15. Schuck P, Perugini MA, Gonzales NR, Howlett GJ, Schubert D (2002) Size-distribution analysis of proteins by analytical ultracentrifugation: Strategies and application to model systems. Biophys J 82(2):1096–1111. 16. Vistica J, et al. (2004) Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal Biochem 326(2):234–256. Fig. S1. Confirmation of MLKL construct expression; N-terminal HA tagging causes loss of MLKL(1–180) potency. (A) Expression of MLKL constructs was induced by dox treatment. (i) Upper panel shows detection of MLKL constructs by the 3H1 anti-MLKL monoclonal antibody; Lower panel shows anti-actin loading control. (ii and iii) Reprobe using anti-FLAG (M2) antibody to detect expression of MLKL constructs lacking the 3H1 epitope. GAPDH (ii) and endogenous MLKL (iii) levels were used as loading controls. (B and C) Two to three biologically independent MDF cell lines derived from Mlkl−/− (B) or wild-type (C) mice were stably infected with a lentivirus encoding dox-inducible N-terminally HA-tagged MLKL(1–180). HA–MLKL(1–180) expression was 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 20 h. Q, Q-VD-OPh; S, Smac-mimetic; T, TNF. PI-permeable cells were quantified using flow cytometry. Hildebrand et al. www.pnas.org/cgi/content/short/1408987111 3 of 8 Fig. S2. Residues required for 4HB domain-mediated killing cluster in two spatial groups: validation of MLKL(1–180) mutant library expression in wild-type and Mlkl−/− MDFs. (A) Three biologically independent MDF cell lines derived from wild-type mice were stably infected with the indicated dox-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. (B) Anti-MLKL Western blot using the 3H1 antibody to detect expression of untagged MLKL(1–180) mutants (as indicated) in each of three biological replicate wild-type (wt1, wt2, and wt3) and Mlkl−/− (ko1, ko2, and ko3) MDF lines. Anti-actin Western blotting was used as a loading control. Hildebrand et al. www.pnas.org/cgi/content/short/1408987111 4 of 8 Fig. S3. Analytical ultracentrifugation analyses of recombinant MLKL(1–169); time course of cell death by MLKL(1–180) corresponds with membrane localization. (A) SV analysis of MLKL(1–169) at three concentrations. (i) Residuals for the c(s) distribution best fits plotted as a function of radial position (cm) from the axis of rotation for MLKL(1–169) at 29 μM (top), 9 μM (middle), and 2.2 μM (bottom). (ii) Normalized continuous size, c(s), distribution is plotted as a function of s20,w for 2.2 μM (solid line), 9 μM (dashed line), and 29 μM (dotted line). Analysis was performed using the program SEDFIT (1) at a resolution of 200, with smin = 0.1, smax = 6, and at a confidence level (F-ratio) = 0.95. Statistics for the nonlinear least squares best fits were as follows: 29 μM, rmsd = 0.007, runs test Z = 45; 9 μM, rmsd = 0.004, runs test Z = 18; and 2.2 μM, rmsd = 0.004, runs test Z = 2. (B) Discrete species analysis of SE data of MLKL(1–169) at 29 μM. Absorbance was measured as a function of radial position from the axis of rotation (cm) once equilibrium was attained at 13,000 rpm (open square) and 20,000 Legend continued on following page Hildebrand et al. www.pnas.org/cgi/content/short/1408987111 5 of 8 rpm (open circle) for MLKL(1–169) at 29 μM. The data were fitted to a discrete species model with mass conservation fitted with two species, as indicated by SV experiments. In this analysis, the monomeric mass was fixed at 20.2 kDa. The fit statistics were as follows: global χr2, 0.9; rmsd, 0.005; runs test Z, 9. Monomer– dimer (dotted line) or monomer–tetramer (dashed line) fits are also shown, which were generated by fixing the species concentrations (ratio of 0.25/0.75, as justified by SV analysis) and masses (monomer, 20.2 kDa; dimer, 40.4 kDa; tetramer, 80.9 kDa). (C) Global analyses of SV and SE. Absorbance measured as a function of radial position from the axis of rotation (cm) for MLKL(1–169) at (i) 29 μM, (ii) 9 μM, and (iii) 2.2 μM at 13,000 rpm (open square) and 20,000 rpm (open circle). (iv) SV data of MLKL(1–169) at an initial concentration of 9 μM [data also fitted to a c(s) distribution in A]. The data in i–iv were fitted to a dimer– trimer self-association model using the program SEDPHAT (2). Statistics for the nonlinear least squares best fits to each experiment were as follows: (i) SE at 29 μM, rmsd = 0.006, runs test Z = 7.7; (ii) SE at 9 μM, rmsd = 0.002, runs test Z = 1.5; (iii) SE at 2.2 μM, rmsd = 0.001, runs test Z = 0.4; and (iv) SV at 9 μM, rmsd = 0.004, runs test Z = 11. (D) Induction of MLKL(1–180) expression and membrane translocation in Mlkl−/− MDFs correlated with the kinetics of cell death. Ectopic expression of full-length MLKL in Mlkl−/− MDFs caused negligible cell death in the absence of TSQ stimulation. Data are mean ± SEM of three biologically independent cell lines assayed in duplicate (n = 6). 1. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78(3):1606–1619. 2. Vistica J, et al. (2004) Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal Biochem 326(2):234–256. Fig. S4. Cluster 1 mutations compromise the capacity of full-length MLKL to induce necroptosis. Biologically independent MDF cell lines derived from wild-type or Mlkl−/− mice were stably infected with a lentivirus encoding dox-inducible mMLKL(1–464) constructs harboring cluster 2 (Y15A/E16A, A, n = 5 wildtype and 5 Mlkl−/− cell lines) or cluster 1 (R105A/D106A, B; E109A/E110A, C; n = 3 wild-type and 3 Mlkl−/− cell lines each) mutations. Expression was induced for 4 h (white bars) or not (black bars), then either left untreated (UT) or treated with the apoptotic (TS) or necroptotic (TSQ) stimulus for 20 h. Q, Q-VD-OPh; S, Smac-mimetic; T, TNF. PI-permeable cells were quantified using flow cytometry. Mutations in either cluster compromise the capacity of full-length MLKL to reconstitute the necroptosis pathway in Mlkl−/− MDFs, and mutations in cluster 1 partially inhibit necroptotic signaling of endogenous MLKL in wild-type MDFs in a dominant-negative manner. (D) Biologically independent cell lines from wild-type or Mlkl−/− mice inducibly express MLKL mutants to equivalent levels. Hildebrand et al. www.pnas.org/cgi/content/short/1408987111 6 of 8 Fig. S5. Mechanistic studies of compound 1 inhibition of MLKL-induced necroptosis. (A) Steady-state binding of compound 1 to immobilized 2xHis6–MLKL (179–464) as measured by SPR. Each point represents the RU level (y axis) at six compound 1 concentrations ranging from 6.25 to 200 μM (x axis). The plateau of this curve demonstrates that compound 1 binding reached saturation. (B) STD–NMR spectra showing nucleotide binding to MLKL. The data show that compound 1 can compete with (i) ATP and (ii) ADP for binding to MLKL pseudokinase domain. The low field region of the off-resonance spectra (purple) shows peaks detected for 200 μM ATP (i) or ADP (ii) in the absence of protein. Peaks marked with asterisks were observed in STD–NMR experiments performed on ATP (i) or ADP (ii) in the presence of 2 μM MLKL(179–464), confirming nucleotide binding. These peaks were diminished in the presence of 200 μM compound 1, confirming that ATP and ADP are displaced from MLKL(179–464) by addition of compound 1. (C) Thermal shift assays demonstrate that Methionine substitution of K219, a crucial ATP binding residue in MLKL, compromises both ATP (red) and compound 1 (green) binding by mMLKL(179–464). These data are consistent with both ATP and compound 1 binding to the ATP binding site of the MLKL pseudokinase domain. Diminished compound 1 binding is indicated by ΔTm values of 2 °C for K219M mMLKL(179–464) in this figure relative to 7 °C for the wild-type counterpart (shown in Fig. 4A). (D) Compound 1 exhibited dosedependent inhibition of necroptotic death by wild-type MDFs stimulated with TSQ (100 ng/mL TNF, 500 nM compound A, and 10 μM QVD-OPh). Toxicity of compound 1 induces death of wild-type MDFs at concentrations ≥5 μM. Mean ± SEM of four independent experiments are shown. (E) Sorafenib, a protein kinase inhibitor with a similar protein kinase target profile to compound 1, did not inhibit TSQ-induced necroptosis in wild-type MDFs at concentrations ≤1 μM. Legend continued on following page Hildebrand et al. www.pnas.org/cgi/content/short/1408987111 7 of 8 Mean ± SEM of triplicate experiments are shown. (F) Radiometric in vitro kinase assay demonstrating that compound 1 has no impact on recombinant RIPK3 kinase activity relative to a DMSO control (0 lanes). Compound 1 concentrations ≥10 μM reproducibly led to enhanced phosphorylation of the MLKL(179–464). Experiment shown is representative of three independent assays. (Left) Dried Coomassie stained 4–12% Bis·Tris gel. (Right) Autoradiograph of the same gel. (G) Two biologically independent MDF cell lines derived from Mlkl−/− mice were stably infected with a dox-inducible wild-type MLKL(1–180) construct. Cells were left untreated or treated with 0.5 or 1 μM of compound 1, 50 μM of Nec-1, or 5 μM of QVD-OPh 1 h before dox induction of wild-type MLKL(1–180) for 20 h. PI-permeable cells were quantified using flow cytometry. A SyproRuby-stained 4-20% Tris-Glycine SDS-PAGE BSA mRIPK3 mMLKL +Compound 1 +DMSO Protein Intensity (sum peptide intensity) B 1E+11 202 PSM 204 PSM 1E+10 1E+09 100000000 10000000 1000000 100000 10000 1000 100 10 1 MLKL +Compound 1 MLKL +DMSO Peptide Intensity (AUC) C D MLKL +Compound mpound 1 MLKL +DMSO Fig. S6. Compound 1 treatment leads to increased phosphorylation of the MLKL activation loop by RIPK3. (A) We resolved, by 4–20% Tris·Glycine reducing SDS/PAGE and stained with SyproRuby, 0.5 μg of mRIPK3 kinase domain reacted with 3.5 μg MLKL(179–464) in in vitro kinase experiments, in the presence of 25 μM compound 1 or DMSO. (B) Comparable amounts of total MLKL peptides (summed peptide intensity) were detected by mass spectometry in compound 1 and DMSO-treated samples. (C) Comparison of MLKL activation loop phosphopeptide intensities (AUC) between compound 1 and DMSO samples. (D) High confidence spectra identifying the doubly phosphorylated (pS345/pS347) tryptic peptide from the activation loop of MLKL. Hildebrand et al. www.pnas.org/cgi/content/short/1408987111 8 of 8