Abstract
As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.
Highlights
Martin Rübbelkea, Dennis Fiegena,b, Margit Bauera, Florian Bindera, James Hamiltona, Jim Kingc, Sven Thammd, Herbert Nara, and Markus Zeeba,1
Structural analysis by NMR spectroscopy was hampered due to significant line broadening in fingerprint spectra and consecutively insufficient quality of nuclear Overhauser enhanced spectroscopy (NOESY) spectra (SI Appendix, Fig. S1)
The addition of IP6 and the detergent NM leads to detachment of the auto-inhibitory α-helix 6, its unfolding, and oligomerization of the four-helix bundle, which is a requirement for its membrane association [19]
Summary
Martin Rübbelkea , Dennis Fiegena,b , Margit Bauera , Florian Bindera, James Hamiltona, Jim Kingc , Sven Thammd , Herbert Nara , and Markus Zeeba,. As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). We present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys to a xanthine class inhibitor. One of the best-studied alternative mechanisms is Tumor Necrosis Factor (TNF)-induced necroptosis, which depends on TNF receptor 1, Receptor-Interacting Protein Kinase 1 (RIPK1), RIPK3, and the mixed-lineage kinase domain-like protein (MLKL) [1]. The first brace helix (α6) plays an important role in the proposed plug-release mechanism [11] It postulates that phosphorylation of MLKL destabilizes the packing of the auto-inhibitory α-helix 6 to the four-helix bundle and, thereby, promotes the detachment of α-helix 6 from the four-helix bundle. The MLKL four-helix bundle is able to oligomerize and integrates into the plasma membrane [13], which leads to permeabilization of the membrane and the breakdown of the cellular structure
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