The goal of our study is to establish the structural basis for Notch receptor-ligand interactions, a critical signaling event for mammalian cell fate determination and the pathogenesis of many cancers. Until recently, we had not been able to “see” how Notch receptors engage ligands Delta-like and Jagged because their nearly undetectable binding affinity prevents reconstitution of stable complexes for structural studies. We overcame this obstacle using directed evolution to engineer high-affinity Delta-like 4 (DLL4) proteins, which stabilized interactions with Notch1 for co-crystallization. The resulting structure revealed a two-site binding interface between epidermal growth factor (EGF) repeats 11 and 12 of Notch1 and the C2 and Delta/Serrate/Lag-2 (DSL) domains of DLL4, respectively. Remarkably, O-linked glycan modifications of Notch1 residues act as surrogate amino acids by making specific and essential contacts with DLL4. It has been established that changes in Notch receptor glycosylation state can bias their recognition towards specific ligands, and the Notch1-DLL4 structure thus rationalizes a mechanism for glycan-mediated tuning of receptor-ligand interactions.
As the Notch1-DLL4 structure represents the only available visualization of a Notch-ligand interface, our current efforts are focused on understanding how Jagged and Delta-like proteins differentially bind and activate Notch1. The Delta-like and Jagged classes of ligands are conserved from flies to humans, and are able to elicit distinct cellular responses despite their shared recognition of cognate Notch receptors. To enable structural and biophysical studies of Notch1 and Jagged1 (Jag1), we have evolved a panel of high-affinity Jag1 variants akin to those we developed to study DLL4. Jag1 variants will first be used to capture and co-crystallize Notch1 and Jag1, though efforts are also underway to determine how biophysical parameters such as mechanical force and ligand ultrastructure contribute to the differential activities of Jag1 vs. DLL4.
Gaining structural access to the Notch-ligand interface was a critical first step toward improved therapeutic targeting of the pathway. With this structural information to guide us, we will now be able to design novel ligands with enhanced selectivity and affinity for a number of immunologically focused applications. For example, we are generating (1) receptor-specific ligands that inhibit tumor growth while minimizing off-target toxicity, (2) stromal cell lines that express high-affinity ligands to enhance T-cell maturation in vitro, and (3) bi-specific ligands that activate Notch on desired cell types. In the future, we hope to create a diverse “toolbox” of engineered ligands for use as diagnostics and therapeutics in a variety of contexts.
Citation Format: Vincent C. Luca, Kevin M. Jude, Nathan W. Pierce, Maxence V. Nachury, Suzanne Fischer, K. Christopher Garcia. Structural basis for Notch1 engagement of Delta-like 4 and Jagged1. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B068.
- ©2016 American Association for Cancer Research.