The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force

Abstract

The Notch signaling pathway relies on a proteolytic cascade to release its transcriptionally active intracellular domain, on force to unfold a protective domain and permit proteolysis, on extracellular domain glycosylation to tune the forces exerted by endocytosed ligands, and on a motley crew of nuclear proteins, chromatin modifiers, ubiquitin ligases, and a few kinases to regulate activity and half-life. Herein we provide a review of recent molecular insights into how Notch signals are triggered and how cell shape affects these events, and we use the new insights to illuminate a few perplexing observations.

Keywords: Notch signaling; mechanism; review; transcription.

Figure 1. Domain schematics and their structure and other structural insights

The central figure shows domain organization of a Notch receptor and a DLL ligand, γ-secretase, and Mib-1. The extracellular domain of Notch receptors (Notch1 shown) consists of multiple iterative EGF repeats followed by the NRR (Negative Regulatory Region), which consists of three LNR (Lin-12 and Notch Repeats) domains and HD (Heterodimerization Domain). EGF repeats for which no structure was determined are colored orange or green (representing the location of Abruptex alleles). EGF repeats in any other color denote recent structure determinations. There is a ~90° bend between EGF5–6, as well as a flexible joint between EGF9–10; mobility near EGF22 is yet to be determined. The intracellular domain of Notch receptors consists of a membrane proximal RAM (RBP-J Associated Molecule) domain, ANK (ankyrin repeats), and a C-terminal TAD (Transactivation Domain) comprised of three NLS (Nuclear Localization Sequences) and degron-containing PEST (for rich in proline, glutamate, serine, and threonine) sequence. The extracellular domain of DSL ligands (DLL4 shown) consists of an N-terminal MNNL (Module at the N-terminus of Notch Ligands) domain, DSL domain, and additional EGF repeats with a potential flexible joint between EGF4–5. (A) X-ray structure of the Notch1-DLL4 complex (PDBID: 4XLW). All domains in the crystal are colored as in the diagram, bound Ca²⁺ ions are shown as yellow spheres, and glycan modifications are shown in a stick representation and colored orange. (B) X-ray structure of Mib1 (PDBID: 4XI6). Mib1 is composed of two N-terminal Mib-Herc2 domains, colored yellow and purple, with a ZZ zinc finger domain (green) in between them. These are followed by two Mib-repeat domain colored orange and red. The Jagged1 N-box peptide bound to the first Mib-Herc2 domain (purple) is shown in a stick representation. The modeled C-box peptide bound to the first Mib-repeat (orange) is also shown in a stick representation with carbon, oxygen, and nitrogen atoms colored yellow, red, and blue, respectively. (C) High resolution CryoEM structure of the γ-secretase complex (PDBID: 5A63). The Nicastrin, Aph-1, PS1, and Pen-2 subunits are colored green, magenta, blue, and yellow, respectively. Note that catalytic aspartates (D257 and D385) in PS1 are not facing the cavity. (D) X-ray structure of Rumi (PDBID: 5F84) modeled with EGF11–13 from Notch1. Rumi is colored light blue and EGF11–13 are colored pink/magenta. Bound UDP moiety is shown in stick representation with carbon, oxygen, and nitrogen atoms colored yellow, red, and blue, respectively.

Figure 2. Regulating transcription

(A) CSL (green) interacts with NICD (dark blue) through the high affinity WFP tri-peptide (hidden by RBPj) and the low affinity ANK domain, positioned at optimal distance by the RAM domain. MAML1 (red) binds to the interface between the ANK and the CSL, and recruits the histone acetylases p300 (pink). P300 convers H3K27 to H3K27ac, a modification enriched in enhancers. Details on specifically how the Notch TAD stimulates transcription are lacking. (B) in the absence of NICD, the short term consequences include recruitment of Kdm5a to the chromatin and loss of H3K4me3. This is a reversible state. (C) If CKII activity is low or absent, Mint/SHARP will instead recruit Kmt2D which will maintain chromatin in the permissive H3K27ac and H3K4me3 state, bookmarking the site for future Notch-mediated activation. (D) in cells lacking NICD and containing active CKII, RBP recruits phosphorylated Mint/SHARP, which bings to NCor. This complex is associated with Histone deacetylases (HDAC) that restore H3K27. The Ezh2 protein in the PRC2 complex than converts H3K27 to H3K27me3, establishing a repressed state propagated further by removal of methyl groups from K4 by Kdm1b.