Deep mutational scanning to characterise MLKL’s killer 4HB domain

• Honours
• Masters by Coursework
• Graduate Research Masters
• PhD

Necroptosis is a lytic form of programmed cell death that is implicated in various inflammatory diseases. Signalling culminates in kinase RIPK3 phosphorylating the final effector protein MLKL, inducing MLKL oligomerisation, translocation to, and permeabilization of the plasma membrane, causing cell death. MLKL’s remarkable transformation from inert monomer to oligomeric, membrane associated killer is enabled by specialised domains: the N-terminal membrane permeabilising 4-helix bundle (4HB), the brace helices which mediate oligomerisation, and the regulatory C-terminal pseudokinase domain. This project will entail Deep Mutational Scanning of MLKL’s killer 4HB domain to identify sequence determinants which regulate its ability to kill cells. Students will develop skills in cloning, library design, mammalian cell culture, screen development and next generation sequencing data analysis.

The Call Lab is in WEHI’s Structural Biology Division, they use a combination of in vivo, cell-based and structural biology methods to answer key questions about immunology. They have recently pioneered the deep mutational scanning workflow at WEHI, enabling in-depth characterisation of the B-cell receptor and COVID-19 proteases. The Murphy Lab is in WEHI’s Inflammation Division. They harness molecular and cell biology as well as biochemical methods to understand the molecular signalling pathways that underpin inflammatory processes. Dr Davies is a senior research officer in the Murphy Lab. Her research focusses on using biochemistry and X-ray crystallography to understand MLKL’s mechanism of action.