Oral Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues (#19)

Katherine A Davies 1 2 , Cheree Fitzgibbon 1 2 , Samuel N Young 1 2 , Sarah E Garnish 1 2 , Wayland Yeung 3 , Diane Coursier 1 2 , Richard W Birkinshaw 1 2 , Jarrod J Sandow 1 2 , Wil I L Lehmann 1 2 , Lung-Yu Liang 1 2 , Isabelle S Lucet 1 2 , James D Chalmers 4 , Wayne M Patrick 5 , Natarajan Kannan 3 , Emma J Petrie 1 2 , Peter E Czabotar 1 2 , James M Murphy 1 2
  1. Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia
  2. WEHI, Parkville, VIC, Australia
  3. Institute of Bioinformatics, University of Georgia, Athens, Georgia, USA
  4. Department of Biochemistry, University of Otago, Dunedin, New Zealand.
  5. School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

Necroptosis is a form of programmed cell death characterized by lack of caspase activity and a loss of plasma membrane integrity. Morphologically similar to necrosis, during necroptosis, the plasma membrane is disrupted, causing release of cellular components to the extracellular fluid and an ensuing inflammatory response. Necroptosis proceeds via a regulated kinase cascade involving Receptor Interacting Protein Kinases RIPK1 and RIPK3. Mixed Lineage Kinase domain-Like protein (MLKL), a pseudokinase, is the final known obligate effector of necroptosis. The MLKL pseudokinase domain is incapable of catalysing phosphotransfer reactions, and is the site of RIPK3 phosphorylation. This phosphorylation event is thought to flip a molecular switch regulated by the pseudokinase domain, resulting in activation of MLKL. Upon activation, MLKL oligomerises, translocates to the plasma membrane, and destabilises it. Details of MLKL’s molecular mechanism of action remain unclear, however, and the literature suggests that there are marked differences in the execution of necroptosis between mouse and human cells.

 

In this study we sought to interrogate how MLKL’s activation mechanism varies between species using structural biology and cell-based approaches1. We solved the crystal structures of the pseudokinase domain of two MLKL orthologues; horse and rat; and tested the ability of nine vertebrate MLKL orthologues to reconstitute necroptosis in human and mouse cell lines. By comparing the new orthologue structures with previously published structures of mouse and human MLKL, we found novel conformations adopted by rat and horse MLKL that may explain the differences in activity observed between species in our cellular assay. We further explored the importance of the novel conformations by mutational analysis. Collectively, our findings suggest that the MLKL:RIPK3 cassette has rapidly co-evolved in different species, potentially due to varying selection pressures exerted by the pathogens that target each organism.

  1. 1. Davies KA, et al. Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues. Nature communications 11, 3060-3060 (2020).