The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Necroptosis is a programmed cell death pathway executed by Mixed Lineage Kinase domain-Like (MLKL).
• MLKL activation, triggered by RIPK3-mediated phosphorylation, involves conformational changes exposing its N-terminal four-helix bundle (4HB) domain.
• The precise mechanisms of MLKL activation, oligomerization, and membrane permeabilization are not fully understood.

Purpose of the Study:

• To investigate the function of the 'brace' helices connecting the 4HB and pseudokinase domains of MLKL.
• To elucidate the role of brace helices in MLKL activation, oligomerization, and cell death signaling.
• To determine the contribution of the pseudokinase domain to MLKL's host specificity.

Main Methods:

• Analysis of MLKL brace helix integrity and its effect on homotrimer assembly and cell death.
• Construction and study of mouse:human MLKL chimeras to map functional domains.
• Assessment of MLKL chimera activity in killing mouse fibroblasts.

Main Results:

• The second brace helix is essential for mouse MLKL homotrimer assembly and necroptotic cell death.
• Brace helices act as a communication device, relaying phosphorylation signals from the pseudokinase to the 4HB domain.
• The first brace helix and adjacent loop are key components of the molecular switch for 4HB activation.
• The pseudokinase domain confers host specificity to MLKL, as demonstrated by mouse pseudokinase domain conferring constitutive killing activity to human MLKL chimeras.

Conclusions:

• MLKL brace helices play an active regulatory role in necroptosis, not merely acting as a structural tether.
• These helices are critical for transmitting activation signals and mediating MLKL's cell death function.
• The pseudokinase domain's role in host specificity highlights its importance in MLKL's biological activity.