BPS2026 – Decoding the role of the ectodomain architecture in native state, activation, and inhibition of the Notch 1 receptor

The Notch signaling pathway is a highly conserved cellular pathway with a major role in intercellular communication during development and adult homeostasis. Dysregulation of the Notch pathway contributes to the progression of developmental diseases, breast cancer, T cell leukemia, and CADASIL. Moreover, Notch is the target of many biotechnological efforts aimed to harness and control its activation. The Notch receptor is a ∼300 kDa transmembrane protein. Its ectodomain includes 36-EGF repeats and the negative regulatory region (NRR) “activation switch” domain. Only three of the 36-EGF repeats are required for ligand binding, leaving a large gap in understanding the role and function of the remaining 33 EGF repeats. Potential flexibility points within the EGF repeats have been previously proposed, begging the question of whether the ectodomain has an extended “rod-like” structure, or a compact structure. Another study indicated potential interactions between the NRR and the EGF repeats, suggesting potential conformational changes necessary for activation. Given the absence of high-resolution structural information, we aim to study the architecture of the Notch ectodomain and its dynamic changes upon ligand binding by measuring distances between light-emitting probes strategically incorporated across the ectodomain. We will report our progress using genetically encoded biosensors that undergo bioluminescence resonance energy transfer (BRET), which reports on distance dependent energy transfer between a donor luciferase and acceptor fluorescent protein. We will present BRET analysis of multiple constructs that contain split luciferase HiBit in the NRR and mNeongreen in different locations throughout the ectodomain.