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.
Characterising the conformation of the HIV envelope susceptible to immune responses
The HIV-1 envelope glycoprotein (Env or (gp120/gp41)3) is expressed on the surface of human immunodeficiency virus (HIV) and at the surface of HIV-infected cells. By default, Env adopts a compact, closed conformation that is not recognized by antibodies inducing the antibody-dependent cellular cytotoxicity (ADCC) response. After engaging with CD4, the glycoprotein at the surface of T-cells that serve as receptor to HIV, Env changes conformations. We have shown that binding of Env to a CD4 mimetic and a specific combination of non-neutralizing antibodies stabilize Env in an intermediate conformation (called State 2A) and that cells expressing Env in this conformation are susceptible to ADCC (Alsahafi et al., Cell Host & Microbe, 2019). The aim of this project is to characterise the state 2A conformation. In this project, we will use single particle cryo-EM and electron-tomography combined with subtomogram averaging to determine the high-resolution 3-dimentional structures of the HIV-1 envelope glycoprotein Env in conformations that are the vulnerable to ADCC. The conformations adopted by Env in the presence of CD4 mimetic, soluble CD4 (full-length and stable domains) and antibodies (full-length or Fab fragments) will be studied and correlated with HIV+ sera mediated ADCC. It is anticipated that the cryo-EM structures will inform the development of new strategies in the treatment and prevention of HIV infections.
Targeting nutrient scavengers to fight bacterial infections
The membrane of bacteria is an important barrier to both keep nutrients, proteins and other essential components inside the cell and toxic compounds outside the cell. Bacteria rely on specific transporters to carry material across its membrane in both direction. Our aim is determine the structure of the transporter, called AdcCB, responsible for transport of zinc a rare but essential metal ion across the membrane of Streptococcus pneumonia. We will determine the structure of AdcCB alone and in complex with two substrate-binding proteins (SBPs), AdcA and AdcAII, using single particle cryo-EM. These studies will inform on the molecular mechanism responsible for selective transport of zinc across the membrane and the role of SBPs in the delivery of the substrate to the transporter. A better understanding of these mechanisms will allow the development of antimicrobial strategies to combat S. pneumoniae infection, the leading cause of bacterial pneumonia that accounts for ~15% of all childhood disease mortalities.
Defining the role of TACAN in pain sensitivity
Mechanosensitive ion channels (MSC) are the sensors for a number of systems including the senses of touch, hearing and balance. They function as mechanotransducers by generating both electrical and ion flux signal as a response to mechanical stimuli. TACAN, is a membrane protein important for detection of painful mechanical stimuli. In this project, you will determine the structures using single particle cryo-EM of TACAN in various lipidic environments. These structures will inform on the molecular mechanism of TACAN in pain sensitivity. The success of this project will make significant contributions to the fundamental understanding of pain sensing and signal sensing by cells in our body. This knowledge will allow the development of novel drugs to treat chronic pain.
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.