Necroptosis is a form of programmed cell death that is activated by signalling from death receptor ligands, such as TNF, and Toll-like receptors. Initiation of necroptosis results in disruption of the inner plasma membrane, cell swelling and release of cellular components that promote inflammation. Key effector proteins in the pathway include receptor interacting protein kinase (RIPK)-1, RIPK3 and the pseudokinase, mixed-lineage kinase domain-like (MLKL). MLKL is the most terminal known component of the necroptotic pathway and contains two functional domains, a N-terminal four-helix bundle (4HB) and a C-terminal pseudokinase domain (PsKD) tethered together by a two-helix “brace” linker. MLKL has retained the ability to bind ATP in the PsKD despite having no catalytic kinase activity. MLKL is activated following phosphorylation of the PsKD by RIPK3, which is thought to induce conformational changes that release the 4HB domain. Subsequently, MLKL is known to oligomerise and translocate to the inner plasma membrane where it destabilises the membrane to promote cell death.
We employed biophysical techniques, including analytical ultracentifugation and size exclusion chromatography, to deduce whether the interaction with ATP regulates MLKL oligomer formation. To understand the structure and dynamics of MLKL activation, we utilized multiple proteomic techniques including hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS) to characterize the changes that occur to MLKL during activation and oligomerisation, and upon ATP binding. Our analysis provided insights into MLKL oligomerisation; a large reorganization of the two-helical “brace” region and protection of several regions in the 4HB and PsKD were observed. Together these results have revealed that MLKL undergoes extensive reorganisation upon activation and oligomerisation, with the changes induced by ATP binding likely reflecting the molecular switch function of MLKL’s pseudokinase domain in controlling cell death by necroptosis.