Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.

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 . 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-molecularweight, membrane-localized complex and cell death. Using alaninescanning 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.pseudoenzyme | RIP kinase | ATP mimetic | programmed necrosis P rogrammed necrosis or "necroptosis" has emerged in the past 5 years as a cell death mechanism that complements the conventional cell death pathway, apoptosis, in multicellular organisms. In contrast to apoptosis, necroptosis does not appear to serve an important role in multicellular organism development (1-3) but participates in the defense against pathogens and is a likely culprit in destructive inflammatory conditions (4-7). Receptor Interacting Protein Kinase-3 (RIPK3) was identified as a key effector of necroptosis in 2009 (4, 5) and its substrate, the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL), in 2012 (8, 9), but the molecular events following RIPK3-mediated phosphorylation of MLKL required to induce cell death are unclear. The RIPK1/ RIPK3/MLKL necrosome has been proposed to activate PGAM5 (phosphoglycerate mutase 5) and Drp1 (Dynamin-related protein 1) to cause mitochondrial fragmentation and cell death (10), but the requirement for PGAM5, Drp1, and mitochondria for necroptosis has been questioned (1, 11-13).We described the structure of mouse MLKL revealing that MLKL contains a C-terminal pseudokinase domain and an N-terminal four-helix bundle (4HB) domain connected by a two-helix linker (the "brace" helices) (1). Based on our mutational and biochemical analyses, we proposed that the catalytically inactive pseudokinase domain functions as a molecular switch and that RIPK3-mediated phosphorylat...

CD1d presents lipid-based antigens (Ag) that are recognised by the semi-invariant T cell receptor (TCR) expressed on Natural Killer T (NKT) cells. While the TCR α-chain is typically invariant, the TCR β-chain expression is more diverse, particularly in mice where at least three different Vβ chains are commonly expressed. We report the structures of Vα14-Vβ8.2 and Vα14-Vβ7 NKT TCRs in complex with CD1d-α-galactosylceramide (α-GalCer), as well as a 2.5 Å structure of the human NKT TCR-CD1d-α-GalCer complex. Both Vβ8.2 and Vβ7 NKT TCRs, as well as the human NKT TCR, ligated CD1d-α-GalCer in a broadly similar manner, thereby highlighting the evolutionarily-conserved nature of this interaction. However, differences within the Vβ domains of the Vβ8.2 and Vβ7 NKT TCR-CD1d complexes not only resulted in altered TCR-β-CD1d-mediated contacts, but also surprisingly modulated recognition mediated by the invariant α-chain. Mutagenesis studies revealed the differing contributions of Vβ8.2 and Vβ7 residues within the CDR2β loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR Vβ usage in NKT cells.