Application of cryo-EM to the study of membrane proteins that are important therapeutic targets
This work will be completed with The University of Melbourne Node.
Targeting viral glycoproteins and bacterial membrane proteins to cure and prevent infection
The current worldwide pandemic reminds us that, despite progress in medicine, infectious diseases remain a serious public health problem with serious economical consequences. Membrane proteins located at the surface of viruses and bacteria are excellent targets to cure and prevent infection. Cryo-EM is the method of choice to determine the structures of membranes, alone and/or in complex with chemical compounds and antibodies at the surface of the infectious agents and/or at the surface of virus-like particles (VLPs) with potentials as vaccines and in drug delivery.
In this project area, we will focus on determining the cryo-EM structures of bacterial and viral glycoproteins that are important therapeutic and prophylactic targets, alone and in complexes. These projects include:
- the cryo-EM structures determination of HIV-1 Env in conformations that are sensitive to attack by antibodies that have potent antibody-dependent cellular cytotoxicity.
- the cryo-EM structures of viral glycoproteins at the surface of VLPs.
- the cryo-EM structures of bacteria transporters such as the ATP-binding cassette (ABC) permeases of Streptococcus pneumonia (AdcCB-AdcA, AdcCBAdcAII).
Structure and mechanism of activation of mechanosensitive ion channels
Ion channels are pore-forming proteins that allow ions to cross cellular membranes. Their function is important for cellular homeostasis and function including pain sensing. Classes of ion channels generate current currents in response to thermal, chemical and mechanical stimuli. Proper function of these channels is critical as they signal the organisms potentially harmful stimuli and allow appropriate reaction to prevent further damage. The loss of the ability of the channel to respond is harmful and is associated with several pathologies. Hyper-activation of this type of ion channels are common causes of chronic pain.
In this project area, we will focus on determining the cryo-EM structures of ion channels, including the recently identified ion channel named TACAN located in sensory neurons. TACAN is the first channel that has been shown to contribute directly to mechanosensitive currents in nociceptors.
Anthrax toxins: threading proteins across cell membranes
Anthrax is bacterial disease that killed 100,000s per year until the 20th century, with still at least 2,000 cases per year nowadays. Inhalation anthrax has mortality rates close to 100% in human, even with treatment. The emergence of antibiotic resistant strains (naturally occurring or developed for bioterrorism) requires new avenues to treat and prevent infection. This project aims to better define the molecular structural features that govern Anthrax intoxication in order to identify new targets for rational treatment. Cryo-EM and biophysical studies will provide essential structural information at early stage and late stage of Anthrax infection. Our studies will provide a detailed understanding of the conformational change that PA loaded with LF undergoes during membrane penetration and how this switch of conformation affects the conformation of LF to maintain translocation ordered. The structures will increase knowledge for transport across membrane and will allow for the design of systems with biotechnological applications, such as for example drugs or toxins delivery. Our study may also reveal fundamental principles on how proteinaceous protein-conduction channels (PPCC) function. The better understanding of these mechanisms will have many applications in biotechnology.
Stopping pore-forming proteins punching holes in membranes
Pore-forming proteins (PFPs) are found throughout life. One of the largest and most sequence diverse PFP superfamilies identified to date are the CDC superfamily of pore-forming toxins. Despite their widespread occurrence and key role in bacterial pathogenesis, the detailed mechanism by which these molecules form pores remains an enigma. We will determine the atomic resolution structures of prepores, pores and intermediate conformations representative of the transition from the prepore to the pore state. Cryo-EM and biophysical data will reveal the role of cholesterol and lipid in driving pre-pore assembly and in triggering conformational change. The structures will shed light on one of the most fundamental biological events (namely, protein insertion into cell membranes), and provide the basis for designing pan CDC inhibitors that might be developed into antibiotics for diverse diseases including gas gangrene, listeria and bacterial pneumonia.
Manipulating signalling in cytokine receptors
Cytokines are small signalling proteins form large multimeric complexes with cell surface receptors leading to broad biological outcomes. Research will focus on determining the structures of two families of cytokine receptors.
Glycoprotein 130 (gp130) is the common signal transducing receptor used by the interleukin (IL)-6 family of cytokines. The IL-11/IL-6 sub-class signals through a gp130 dimer; while leukemia inhibitory factor (LIF) and oncostatin M (OSM) use gp130 in complex with a co-receptor. Efforts to understand the process of transmission of the message across the cell membrane have been hampered by a lack of structural information. We have determined the structure of the complete extracellular domains of the IL11 signalling complex by cryo-electron microscopy. This project aims to determine the structures of IL-6 family cytokines in complex with their intact receptors, including intracellular regions bound to JAK kinases, in order to elucidate the molecular mechanisms of signal transduction. This knowledge will identify new approaches to investigate cytokine biology in general, and provide a structural platform that will enable researchers to understand dysregulated cytokine signalling in disease. It is anticipated that cryo-EM structures of these signalling complexes at high resolution will provide the framework to identify new target-based approaches to inhibit signalling. Inhibitors of IL-6 family cytokine signalling may provide potential therapeutics for a range of cancers and other conditions such as cardiovascular fibrosis.
The betacommon family of receptors include the cytokines IL-3, IL-5 and GM-CSF that regulate the survival, proliferation, differentiation and activation of haematopoietic cells. We have determined the structures of the extracellular regions of the GM-CSF and IL-3 receptor binary and ternary complexes but structures of the transmembrane and intracellular regions bound to signalling molecules such as the JAK kinases are unknown. Our aim is to determine the structures of the intact receptors by cryo-EM as a basis for understanding transmembrane signalling. We expect that single small molecule inhibitors of GM-CSF/IL-3/IL-5 cytokine receptor assembly can be discovered based on the structures which could simultaneously inhibit the action of the three cytokines. Such inhibitors could be developed into drugs to treat certain forms of leukaemia, inflammatory diseases and asthma.
Understanding key drug targets in Alzheimer’s disease
In this program of work the overall aim is to determine the structures of key proteins in Alzheimer’s disease as a basis for understanding their normal physiological function and to guide structure-based drug discovery. In the late nineties we embarked on an ambitious project to determine the complete structure of amyloid precursor protein (APP), a membrane-bound receptor that plays a central role in Alzheimer’s disease (AD). APP contains the Abeta peptide thought to cause the disease. We have determined the structures of a number of components by crystallography and now aim to visualise the complete intact structure of APP by cryo-EM. Anti-Abeta monoclonal antibodies are the leading therapeutics being tested in human clinical trials. My lab has visualised how three such clinical antibodies recognise the Abeta peptide. We now aim to visualise how some of these antibodies bind the Abeta fibrils, one of the principal pathologies in AD.