Structural features of the Notch ankyrin domain-Deltex WWE2 domain heterodimer determined by NMR spectroscopy and functional implications

• Published in: Structure (London) 2023-05, Vol.31 (5), p.584-594.e5
• Main Authors: Carter, Andrea A., Ramsey, Kristen M., Hatem, Christine L., Sherry, Kathryn P., Majumdar, Ananya, Barrick, Doug
• Format: Article
• Language: English
• Subjects: ankyrin repeats; Deltex; NMR; Notch; Notch signaling; protein-protein interactions; RING domain; ubiquitin ligase; WWE2 motifs
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2023.03.003

The Notch signaling pathway, an important cell fate determination pathway, is modulated by the ubiquitin ligase Deltex. Here, we investigate the structural basis for Deltex-Notch interaction. We used nuclear magnetic resonance (NMR) spectroscopy to assign the backbone of the Drosophila Deltex WWE2 domain and mapped the binding site of the Notch ankyrin (ANK) domain to the N-terminal WWEA motif. Using cultured Drosophila S2R+ cells, we find that point substitutions within the ANK-binding surface of Deltex disrupt Deltex-mediated enhancement of Notch transcriptional activation and disrupt ANK binding in cells and in vitro. Likewise, ANK substitutions that disrupt Notch-Deltex heterodimer formation in vitro block disrupt Deltex-mediated stimulation of Notch transcription activation and diminish interaction with full-length Deltex in cells. Surprisingly, the Deltex-Notch intracellular domain (NICD) interaction is not disrupted by deletion of the Deltex WWE2 domain, suggesting a secondary Notch-Deltex interaction. These results show the importance of the WWEA:ANK interaction in enhancing Notch signaling. [Display omitted] •We have made backbone NMR assignments of the tandem WWE2 domain of Drosophila Deltex•By measuring chemical shift perturbations, we mapped Notch ANK-binding site to WWEA•Mutating binding site residues disrupts Deltex-mediated transcriptional enhancement Notch signaling determines cell fate in animals. Notch receptor activity is modulated by several effectors, including Deltex, an E3 ubiquitin ligase. Here, Carter et al. use NMR to map the Notch binding site on the Deltex WWE2 domain and show this interaction to enhance Notch-mediated transcriptional activation.