Conserved residues at a Notch1 crystal packing interface. (A) The Notch1 NRR and a symmetry mate are depicted in molecular surface representation. The LNR domains are colored in light and dark pink and the HD domains colored in light and dark cyan in molecules 1 and 2, respectively. (B) Molecule one surface after a counterclockwise 90-degree rotation about the axis shown. In this view, the surface-exposed face of LNR-C is facing outward. Amino acid residues involved in the crystal contact interface are colored purple. (C) Surface of the Notch1 NRR colored according to amino acid conservation using the scale shown (as described in the legend for Figure 1B). (D) Surface of the Notch1 NRR illustrating the 6 crystal contact residues that have been mutated to Ala (hot pink). (E) Effect of mutating the LNR-C crystal contact interface in Notch reporter gene assays. Signaling in coculture assays with NIH 3T3 cells alone or stably expressing the ligand Jagged-2 was examined using chimeric full-length receptors in which the RAM and ANK domains of NOTCH1 were replaced with the DNA-binding domain of the transcription factor GAL4 (“Luciferase reporter/urea sensitivity assays”). Firefly luciferase activity was normalized to the internal Renilla control and expressed relative to the reporter gene activity of the unmutated chimeric receptor cocultured with NIH 3T3 cells alone. Error bars represent the SE of the 3 replicate measurements made for each experimental condition.