Crystal structure, biochemical and biophysical characterisation of NHR1 domain of E3 Ubiquitin ligase neutralized

Notch signaling controls diverse developmental decisions of central importance to cell activity. One of the conserved positive regulators of Notch signaling is Neuralized, the E3 Ubiquitin ligase enzyme that regulates signaling activity by endocytosis. Neuralized has two novel repeats, NHR1 and NHR2, with a RING finger motif at the C-terminus. Both endocytosis of the Notch ligand, Delta, and inhibition of Notch signaling by Tom, a bearded family member, require the NHR1 domain. Here we describe the first crystal structure of NHR1 domain from Drosophila melanogaster, solved to 2.1 A resolution by X-ray analysis. Using NMR and other biophysical techniques we define a minimal binding region of Tom, consisting of 12 residues, which interacts with NHR1 and show by interfacial analysis of protein monolayers that NHR1 binds PI4P. Taken together, the studies provide insight into molecular interactions that are important for Notch signaling.