X-ray structure of the human NOTCH2 negative regulatory region (NRR) in its autoinhibited conformation, and models for signal activation. The top panel shows a ribbon representation of the NOTCH2 NRR. The LNR modules are colored in different shades of pink and purple, and the HD is in white and green. The three bound Ca²⁺ ions are shown in green, the bound Zn²⁺ ion is purple, and the disulfide bonds are red. The positions of S1 and S2 cleavage are indicated with red arrows. The bottom panel shows the model for activation by mechanical force driven by the DSL endocytosis. For unbound Notch, the LNRs (pink structure in 1–4) protect the S2 cleavage site in HD (white structure in 1–4). When Notch binds to DSL, which then undergoes endocytosis (1 and 2), this generates a mechanical force for disengaging LNRs from the HD (shown in 1–3). This relaxation between LNRs–HD interaction allows the metalloprotease to access the S2 cleavage site (shown in 4), leading to Notch activation. The figure is adapted from Gordon et al. (2007, 2008).