Necroptosis is a form of cell death where the inner plasma membrane is compromised, resulting in cell swelling and the release of cellular contents that invites an inflammatory immune response.
The core pathway controlling necroptosis is best described downstream of the TNF receptor, where the conventional protein kinases, Receptor Interacting Protein Kinase (RIPK)-1 and RIPK-3, regulate the activation of the terminal effector, the pseudokinase Mixed Lineage Kinase-domain Like (MLKL). Phosphorylated and oligomerised MLKL can be found at the inner plasma membrane of necroptotic cells, however it is not clear how this leads to the break down of the cell wall.
MLKL has two functional domains, an N-terminal four-helix bundle (4HB) domain and a C-terminal pseudokinase domain (PsKD), which are tethered by a two-helix linker. Our group has shown that it is the 4HB domain that executes cell death, and propose that the PsKD functions as a molecular switch, whereby phosphorylation by RIPK3 results in a conformational change and the release of the 4HB domain.
Like many pseudokinase domains, that of MLKL has retained the ability to bind to ATP but lacks the essential residues that confer catalytic activity. This raises questions around whether this is merely an evolutionary consequence of retaining a kinase-like fold or if ATP is important to the killing function of MLKL. We have used a variety of biophysical techniques, including Hydrogen-Deuterium Exchange (HDX) Mass Spectrometry, Analytical Ultracentrifugation (AUC), Small Angle X-ray Scattering (SAXs) and analytical Size Exclusion Chromatography (SEC) to examine the molecular switch mechanism underlying MLKL activation and oligomerisation, and how this can be impacted by ATP-binding.