STRUCTURE-INFORMED STUDIES OF THE EXTRACELLULAR REGION OF THE HUMAN NOTCH RECEPTOR IN HEALTH & DISEASE

The Notch pathway is an essential signal transduction system which regulates crucial decisions in cell biology. These are vital for embryonic development and for the maintenance of adult tissue. In its simplest form, a cell-surface expressed ligand activates the pathway by binding to the Notch receptor protein presented on the outside of a neighbouring cell. This leads to cleavage events such that the intracellular portion of the receptor is released and travels to the nucleus where it forms a complex with other proteins to activate specific genes responsible for regulating cell behaviour. Abnormal loss or gain of Notch activity is associated with many human developmental disorders, adult onset diseases and cancers, making it a key target for therapeutic intervention. Despite extensive study of the downstream consequences of activation, many aspects of the early events leading to Notch cleavage and generation of the signal remain unclear. This is because we lack understanding of the overall shape of Notch and the way in which it can interact with its ligands. Because it is such a large protein of unknown flexibility, with a fibrous rather than globular organisation, it is not technically feasible to study Notch in its intact, full-length state. Instead, due to its modular construction it can be divided into different regions based on functional importance.

In previous work, we have established a structural model for the EGF4-13 polypeptide which contains a previously unrecognized region of flexibility between EGF9 and EGF10. We now wish to extend our structural and functional studies to EGF20-27 and the domains (EGF14-20) that link this to the ligand-binding region. We are keen to understand whether domains within EGF20-27 which incorporate a region known as Abruptex (Ax) can interact intra or intermolecularly with the ligand-binding region (LBR).

This will enable us to understand the overall shape of the extracellular domain of Notch from EGF4 to EGF27, to identify regions that are rigid and those that are flexible, to understand the significance of the Ax/LBR interaction for structure, function and regulation of Notch activity at the cell surface. Fundamental understanding of how the Notch signal is generated should allow us to develop reagents to manipulate Notch activity and facilitate advances in many aspects of cell biology, including stem cell biology. In addition, since dysfunction of the pathway results in many inherited and acquired forms of disease, such as cancer, these reagents are likely to have therapeutic potential.

Understanding the molecular basis of Notch signaling is of significant biomedical importance. Despite the identification of the Notch receptor more than 30 years ago, we lack a description of its full structure, which is crucial for understanding how it functions at the cell surface. This major conceptual gap will be addressed by analyzing the structure and dynamics of an important region in its extracellular domain. Our previous work established a structural model of EGF4-13. Strikingly, we observed that the receptor does not simply extend from the cell-surface, but has the potential to adopt a range of dynamic conformations. We now aim to address the structure and function of EGF20-27 and the region that links this to the ligand-binding region (LBR) studied previously.

A multidisciplinary approach will be taken:

1. To study the structure & dynamics of a range of EGF domain fragments from the EGF14-27 region of the Notch receptor using mainly NMR spectroscopy, calcium-binding measurements & SAXS to define the overall architecture of this critical region.
2. To further characterise the binding interface of the Ax/EGF11-13 (LBR) interaction using site-directed mutagenesis and established FACS and ELISA-type assays, to determine if it confers an intramolecular hairpin loop structure to Notch and whether or not ligand competes with Ax.
3. To perform Notch activation experiments in cells utilizing structure-informed site-directed mutants of full-length Notch and purified or cell-surface-expressed ligands to test the functional importance of the Ax/LBR pairwise interaction and EGF domain interface rigidity/flexibility for activity.

These data will enable us to understand the cell-surface architecture of Notch from EGF4 to EGF27, inform on the biological significance of the Ax/LBR interaction for structure and function, and give new insights into the molecular basis of activatory and inhibitory Notch complexes at the cell surface.