Journal article

Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis

Sarah E Garnish, Yanxiang Meng, Akiko Koide, Jarrod J Sandow, Eric Denbaum, Annette V Jacobsen, Wayland Yeung, Andre L Samson, Christopher R Horne, Cheree Fitzgibbon, Samuel N Young, Phoebe PC Smith, Andrew I Webb, Emma J Petrie, Joanne M Hildebrand, Natarajan Kannan, Peter E Czabotar, Shohei Koide, James M Murphy



Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulati..

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Awarded by National Health and Medical Research Council

Awarded by NIH

Funding Acknowledgements

We thank staff at the Australian Synchrotron MX beamline for assistance with data collection. This research was undertaken in part using the MX2 crystallography beamline at the Australian Synchrotron, Victoria, Australia, and made use of the ACRF Detector. We acknowledge scholarship support for S.E.G. and A.V.J. (Australian Government Research Training Program Stipend Scholarships), S.E.G. (Wendy Dowsett Scholarship), Y.M. (Melbourne Research Scholarship and AINSE PGRA scholarship). We are grateful to the National Health and Medical Research Council for fellowship (J.M.H., 1142669; P.E.C., 1079700; J.M.M., 1105754, 1172929), grant (1057905; 1124735, 2002965) and infrastructure (IRIISS 9000653) support; Australian Cancer Research Foundation; and the Victorian Government Operational Infrastructure Support scheme. Funding for N.K. from NIH (5R01GM114409 and 1R35GM139656) is acknowledged.