Protein interactions during cell death by necroptosis

Protein interactions during cell death by necroptosis

A new molecular 'freeze frame' technique has allowed the researchers to see key steps in how the protein mixed lineage kinase domain-like (MLKL) kills cells.

Small proteins called 'monobodies' were used to freeze MLKL at different stages as it moved from a dormant to an activated state, a key process that enables an inflammatory form of cell death called necroptosis. The team were able to map how the three-dimensional structure of MLKL changed, revealing potential target sites that might be targets for drugs - a potential new approach to blocking necroptosis as a treatment for inflammatory disease.

MLKL is a key protein in necroptosis, being the 'executioner' that kills cells by making irreparable holes in their exterior cell membrane. This allows the cell contents to leak out and triggers inflammation - alerting nearby cells to a threat, such as an infection.

The author said MLKL was activated within a protein complex called a 'necrosome' which responded to external signals.

"While we know which proteins activate MLKL, and that this involves protein phosphorylation, nobody had been able to observe any detail about how this changes MLKL at the structural level. It happens so fast that it's essentially a 'molecular blur'," the author said.

A new technology - monobodies - developed by the team, was key to revealing how MLKL changed.

Monobodies that specifically bound to different 'shapes' of MLKL were used to capture these within cells, another author said.

"These monobodies prevented MLKL from moving out of these shapes - so we could freeze MLKL into its different shapes," the author said.

"We then used structural biology to generate three-dimensional maps of these shapes which could be compared. This revealed that MLKL passed through distinct shape changes as it transitioned from being activated through to breaking the cell membrane."

The senior author said the structures provided the first formal evidence for how MLKL changed its shape after it was activated.

"Until now, we've speculated that this happens, but it was only with monobodies that we could actually prove there are distinct steps in MLKL activation," the author said.

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  receptor-interacting serine/threonine protein kinase (RIPK3) following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3.

"Necroptosis is an important contributor to inflammatory conditions such as inflammatory bowel disease. There is intense interest in MLKL as a key regulator of necroptosis - and how it could be blocked by drugs as a potential new anti-inflammatory therapy."