Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Application of cryo-EM to understand the molecular structure and signal transduction of the Ephrin Receptor Pseudokinase EphA10 (#125)

Hai-Tian Chen 1 2 3 , Joshua M Hardy 1 2 3 , Marija Dramićanin 4 , Lung-Yu Liang 1 2 , Andrew Leis 2 5 , Onisha Patel 2 , Isabelle S Lucet 1 2 3
  1. ACRF Chemical Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC , Australia
  2. Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia
  3. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Parkville, VIC, Australia
  4. Protein Production Facility, Walter and Eliza Hall Institute of Medical Research, Bundoora, VIC, Australia
  5. Structural Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

Ephrin receptors (EphRs) are membrane receptors which transmit signals across the membrane after recognition of membrane-tethered ephrin ligands. The intracellular tyrosine kinase domain of active EphR can phosphorylate tyrosine residues upon ligand-dependent oligomerization and receptor activation. Two EphRs, EphA10 and EphB6, are pseudokinases that cannot catalyse phosphorylation but can still interact with other proteins. Upregulation of EphA10 is often identified in cancers such as triple-negative breast cancer and prostate cancer suggesting that the non-catalytic functions of EphA10 can be oncogenic. Our lab has previously demonstrated that EphA10 can still bind to ATP and that EphB6 is a substrate for active EphB41. These findings suggest that the EphR pseudokinases may be conformational switches, allosteric regulators for active EphR or a hub for the assembly of signalling complexes. Nevertheless, the exact mechanism of EphA10 activation, oligomerization and the downstream signalling cascade remains poorly understood.

To date, we have expressed and purified the ectodomains of EphA10 and ephrin A3/A4/A5 ligands in insect cells. We have determined a low-resolution reconstruction of the EphA10/ephrin-A3 complex using cryo-electron microscopy (cryo-EM). Due to the high flexibility of the EphA10 ectodomain, we are developing stabilising interactors (nanobodies/small molecules) to improve the stability and order of the complex. To understand the structure and function of full-length EphA10, we have established mammalian cell expression systems. Additionally, we are performing fluorescence live cell imaging using confocal and lattice light-sheet microscopy to screen small molecule inhibitors and investigate EphR-mediated cell-to-cell communication.

Together, these approaches will provide insight into EphA10 function and guide the development of novel cancer therapies.

  1. Liang, L., Roy, M., Horne, C., Sandow, J., Surudoi, M., Dagley, L., Young, S., Dite, T., Babon, J., Janes, P., Patel, O., Murphy, J. and Lucet, I., (2021) “The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs”, Biochemical Journal, 478(17), 3351-3371.