A soluble prepore state of the perforin-like macrophage immune effector (2.8 Å cryoEM reconstruction; false coloured)
“One of the most exciting things for me is being able to visualise our immune effectors, our pore-forming proteins at atomic resolution. That’s something that I never thought would be possible in the course of the Centre – or actually in the course of our scientific career. We have gone from seeing very blurry shapes of these molecules to seeing them in exquisite detail.”
This work uses structural biology and biochemistry to understand how pore-forming immune effectors function to destroy targets. In particular, we are interested in three mammalian proteins: Complement component-9 (C9), Macrophage Expressed Gene-1 (MPEG-1) and Perforin. These three molecules play key roles in the destruction of pathogenic microbes, virally infected cells and malignant cells. Concomitant with working on these protein complexes, we are also developing new and better approaches for sample preparation for cryogenic electron microscopy experiments and for determining protein structures in the context of intact, cryogenically preserved cells.
PROF. JAMES WHISSTOCK
AT A GLANCE
Over the past seven years of the Centre, we have completed our ambition to understand the basic mechanism of how perforin-like immune effectors form pores, with high-resolution structures now determined for all of the key intermediates that perforin-like proteins progress to pore formation, as well as the structure of the pore itself. During 2020, this work was naturally impacted as our team pivoted to focus on the COVID-19 pandemic. Despite this, we made excellent progress on understanding the kinetics of pore formation, and in gaining a molecular understanding of how these proteins interact with lipid membranes. This work takes advantage of the single-molecule imaging technologies developed by Kat Gaus and her team. Collectively this work is now nearing completion and will be completed and published in 2021. The next challenge, which is well underway, is to use this wealth of information in the development of therapeutic molecules that inhibit the three major human pore-forming immune effectors: perforin, the terminal proteins of Complement, and Macrophage Expressed Gene-1. Future work on these effectors will be supported through funding from the NHMRC NHMRC Ideas Grant (led by Associate Investigator Dr. Ruby Law).
Regarding the technology developed under this theme, the Centre electron microscopy initiative continues to expand and gain pace. The Monash Ramaciotti Centre for Electron Microscopy was established with the support of the Centre and will continue to grow in the future. The infrastructure is now supported through NCRIS (via Microscopy Australia), and Centre members such as A/Prof. Alex de Marco and A/Prof. Georg Ramm continue to develop their vision through the support of academic appointments and initiatives such as the Chan Zuckerberg Initiative, respectively.
Human perforin-like immune effectors visualised at atomic resolution in multiple states throughout the pathway of pore formation.
- Developed an extensive structure-based understanding of how perforin-like proteins form pores and how this process is controlled by factors such as acidification or proteolysis.
- Developed automation techniques
- for cryo-lamella production to permit
- in situ structural biology.
- Developed software and instrumentation to drive in situ structural biology.
The Monash Ramaciotti Centre will expand to include new microscopes that will arrive in early
2022; these will include the state-of-the-art 300 KeV TEM microscope. This infrastructure will ensure that Australian scientists continue to be supported at the cutting-edge of molecular imaging.