Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Two novel orthologue MLKL pseudokinase structures identify structural features important for necroptosis (#148)

Katherine Anne A Davies 1 , Sam N Young 1 2 , Emma J Petrie 1 2 , James M Murphy 1 2 , Peter E Czabotar 1 2
  1. The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia

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, including activation, oligomerisation and how it interacts with the plasma membrane, remain unknown.

Both mouse and human MLKL have been used to study necroptosis in cells and in vitro, however, differences in killing activity, membrane permeabilisation1 and oligomerisation2, 3 have been reported between them. We recently performed a cell based study on chimeric mouse and human MLKL fusion proteins, and were able to define key determinants of killing ability for each species3. In our new study, we have sought to further understand the key structural and sequential features of MLKL that allow it to execute necroptosis. To do so, we have examined the ability of MLKL orthologues to reconstitute necroptosis in human and mouse cell lines. We have also solved two novel crystal structures; the pseudokinase domain of rat and horse MLKL. Contrasted with the existing mouse and human pseudokinase structures, these new structures highlight structural variances that may explain the functional differences between MLKL orthologues.

  1. Tanzer MC, Matti I, Hildebrand JM, Young SN, Wardak A, Tripaydonis A, Petrie EJ, Mildenhall AL, Vaux DL, Vince JE et al. (2016) Evolutionary divergence of the necroptosis effector MLKL. Cell death and differentiation. 23(7):1185-1197.
  2. Petrie EJ, Sandow JJ, Jacobsen AV, Smith BJ, Griffin MDW, Lucet IS, Dai W, Young SN, Tanzer MC, Wardak A et al. (2018) Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis. Nature communications. 9(1):2422.
  3. Davies KA, Tanzer MC, Griffin MDW, Mok YF, Young SN, Qin R, Petrie EJ, Czabotar PE, Silke J, Murphy JM. (2018) The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis. Cell death and differentiation.