Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Necroptosis is a regulated cell death pathway implicated in various diseases.
• Tumor necrosis factor (TNF) activates necroptosis through RIPK1/RIPK3 kinases and the mixed-lineage kinase domain-like protein (MLKL).
• Activated MLKL oligomerizes and inserts into the plasma membrane via its executioner domain, leading to cell death.

Purpose of the Study:

• To elucidate the structural basis of MLKL inhibition by xanthine class inhibitors.
• To investigate the mechanism by which these inhibitors prevent MLKL activation and oligomerization.
• To compare the mechanism of action of xanthine inhibitors with other known MLKL inhibitors like Necrosulfonamide.

Main Methods:

• X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy to determine the co-structures of the human MLKL executioner domain bound to a xanthine inhibitor.
• NMR-based functional assays to assess the compound's effect on MLKL conformation and the role of specific residues (e.g., Phe148).
• Real-time and diffusion NMR studies to monitor MLKL oligomerization in the presence of xanthine derivatives.

Main Results:

• The co-structures revealed that the xanthine inhibitor covalently binds to Cys86 and stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by interacting with Phe148.
• An NMR assay confirmed that mutations at Phe148 (F148A) render MLKL unresponsive to the inhibitor, highlighting the importance of this interaction.
• NMR studies demonstrated that xanthine derivatives effectively inhibit MLKL oligomerization.
• The study suggests that Necrosulfonamide, another Cys86-targeting MLKL inhibitor, operates via a distinct mechanism.

Conclusions:

• Xanthine class inhibitors bind to the MLKL executioner domain, stabilizing an auto-inhibitory conformation and preventing oligomerization.
• The interaction with Phe148 is crucial for the inhibitory activity of xanthine derivatives.
• These findings offer a detailed molecular understanding of MLKL inhibition, potentially aiding in the development of novel necroptosis-targeting therapeutics.
• The distinct mechanisms of action between xanthine inhibitors and Necrosulfonamide provide avenues for developing combination therapies or inhibitors with specific targeting profiles.