Protein kinases are essential regulators of eukaryotic cellular signalling and mutations within these domains are a common cause of carcinogenesis in humans. Only recently have related proteins, termed pseudokinases, emerged as crucial modulators of signal transduction, with mutations in several pseudokinases likewise implicated in human diseases. Although these domains have historically been viewed as the catalytically-dead 'poor cousins' of active protein kinases, pseudokinases are found throughout evolution, with examples now identified in bacteria, yeast, plants, apicomplexan parasites, worms and vertebrates: illustrating that pseudokinases serve essential, evolutionarily-conserved signaling functions and are not merely cellular junk.

In 2012, one such pseudokinase, Mixed lineage kinase domain-like (MLKL), was identified as a component of the ‘necrosome’, the multi-protein complex that triggers tumour necrosis factor (TNF)-induced cell death by the process termed necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. While 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, we show that 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 we show that its essential non-enzymatic role in necroptotic signaling is induced following phosphorylation by the upstream bona fide protein kinase, RIPK3. Remarkably, 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. These studies illustrate that MLKL serves as an essential checkpoint in the necroptosis cell death pathway and form the basis for therapeutic targeting of MLKL as a novel avenue to counter prevalent inflammatory diseases, such as psoriasis or arthritis.