Domain organization of the Notch pathway receptors, ligands and co-ligands from fly, worm and mammals. A) Notch receptors are large Type I proteins that contain multiple extracellular EGF-like repeats. The single Drosophila (dNotch) and 4 mammalian Notch receptors (mNotch1–4) differ in the number of repeats (29–36) but all are much longer than the C. elegans Notch proteins (cLIN-12 and cGLP-1). Repeats 11–12 (orange) and 24–29 (green) mediate interactions with ligands. EGF repeats may contain consensus motifs for fucosylation by O-Fut1 and glycosylation by Rumi; the putative distribution of shared (green) and unique fucosylation (Cyan) and glycosylation (magenta) sites are shown for mNotch1 and mNotch2. Note that the ligand binding regions differ in their modification patterns. EGF repeats are followed by the Negative Regulatory Region (NRR), which is composed of three cysteine-rich Lin12-Notch repeats (LNR-A, B and C) and a heterodimerization domain (HD). In contrast to Drosophila Notch (dNotch), mammalian Notch proteins are cleaved by furin-like convertases at site 1 (S1). See text for more details on the intracellular domain. B) Ligands and potential ligands of Notch receptors can be divided into several groups based on their domain composition. Classical DSL ligands contain DSL, DOS and EGF motifs, and are not found in C. elegans (DSL/DOS/EGF ligands). C. elegans and mammalian DSL-only ligands lacking the DOS motif (DSL/EGF ligands) are a subtype of DSL ligands that may act alone (e.g., mDLL4) or in combination with DOS co-ligands (e.g., cDSL-1, and perhaps Dll3). This sub-family includes diffusible ligands. Functionally tested DOS co-ligands are marked with an asterisk; the role of mammalian DOS proteins is yet to be explored. Non-canonical ligands lack DSL and DOS domains but may act to facilitate the activation of Notch by DSL ligands and/or DOS co-ligands. Red brackets mark domains that have been crystallized alone or in combination with binding partners; some structural details will be addressed here but see (Blacklow upcoming review JCI). C) Details of the mouse Notch1 TMD (boxed) and flanking residues showing the cleavage sites and corresponding products. After ligand binding, Notch is cleaved at S2 by metalloproteases. γ-secretase can cleave multiple scissile bonds at S3 but only NICD molecules initiating at Val (V1744) evade N-end rule degradation (NICD-V). Cleavage then proceeds towards S4 until the short Nβ peptides can escape the lipid bilayer; most Nβ peptides are 21 amino acids long. The V1744G and K1749R amino acid substitutions (colorized) shift the S3 cleavage site (see text for details).

The Core Notch Signaling Pathway is mediated by regulated proteolysis. Upon translation, the Notch protein is glycosylated by O-fut and Rumi, which are essential for the production of a functional receptor. The mature receptor is produced after proteolytic cleavage by PC5/furin at Site 1 (S1) and thereafter targeted to the cell surface as a heterodimer held together by non-covalent interactions. In cells expressing Fringe, the O-fucose is extended by the glycosyltransferase activity of Fringe, altering the ability of specific ligands to activate Notch. The Notch receptor is activated by binding to a ligand presented by a neighboring cell. Endocytosis and membrane trafficking regulate ligand and receptor availability at the cell surface. Ligand endocytosis is also thought to generate sufficient force to promote a conformational change that exposes Notch to cleavage at site S2 by ADAM metalloproteases (perhaps following heterodimer dissociation at S1). Juxtamembrane cleavage at S2 generates the membrane-anchored NEXT (Notch extracellular truncation) fragment, which is a subtrate for the γ-secretase complex. γ-secretase cleaves the Notch TMD progressively (from site S3 to S4; see Figure 1C) to release NICD (Notch intracellular domain) and Nβ peptides. γ-secretase cleavage can occur at the cell surface or in endosomal compartments however cleavage at the membrane favors the production of more stable form of NICD (see text for details). In the absence of NICD, the DNA-binding protein CSL associates with ubiquitous corepressor (Co-R) proteins and histone deacetylases (HDACs) to repress transcription of target genes. When NICD enters the nucleus, its binding to CSL may trigger an allosteric change that facilitates displacement of transcriptional repressors. Mastermind (MAM) then recognizes the NICD/CSL interface, and this tri-protein complex recruits coactivators (Co-A) to activate transcription.

Atomic resolution details of the ligand-binding domain (EGF 11–13) from human Notch1 (PDB:2VJ3), the NRR from human Notch2 (PDB:2OO4) and the Notch-binding domain from human Jagged1 (PDB:2VJ2) generated with MacPymol (http://www.pymol.org). A) The ligand-binding domain of Notch1 (schematically depicted to the left) is centered on EGF repeat 12. The essential amino acids that coordinate Ca⁺⁺ binding are shown in blue. O-glycosylation (Ser458, Ser496) and O-fucosylation (Thr466) sites are shown. Of note, the equivalent mutation to Glu455Val abolishes ligand binding in Drosophila, and this interface in hNotch1 was suggested to interact with hJagged1 DSL based on in silico docking models (Cordle et al., 2008a). However, glycosylation on Ser458 may block access to this site. Thr466 is essential for productive Notch activation in the mouse but not in the fly. B) The NRR domain folds to protect the S2 cleavage site (colorized side chains), which is located in a pocket protected by LNR-A, the HD-C helix, and the LNR-B/A linker. The furin cleavage site S1 lies within an unstructured loop that was removed to facilitate crystallization. LNR repeats bind calcium; chelation of these Ca⁺⁺ atoms lead to NRR dissociation and Notch activation. See text and (Gordon et al., 2007) for further details. C) The crystal structure of the DSL, DOS and EGF repeat 3 of hJagged1 (schematically depicted to the right in trans binding orientation), highlighting the putative Notch binding interface (facing left)). The DSL fold is distinct from the EGF fold; amino acids in DSL that were shown to be required for interaction with Notch are labeled in red (see (Cordle et al., 2008a) for detail). Phe207Ala substitution generates a null protein whereas Arg203Ala and Phe199Ala substitutions ablate trans but not cis binding. Asp205Ala and Arg201Ala are hypomorphic. The DOS domain contains two conserved, atypical EGF repeats (defined by the presence of the conserved amino acids shown in blue (Komatsu et al., 2008)). Tyr255 is characteristic of Jagged DSL ligands and is replaced by a small hydrophobic amino acid in Delta-like proteins; this residue may be involved in defining sensitivity to Fringe.

Mapping known autosomal dominant mutations on the surface of hJagged1 indicates that the DOS domain could form part of the Notch-binding interface. The hJagged1 ribbon structure (A) was surface rendered and rotated such that the Notch-binding interface is facing the reader (B–E). B) Structural model of the wild-type ligand showing biologically relevant residues. Alagille syndrome (ALGS)-associated missense mutations that are likely to affect disulfide bonding (and thus the structural integrity of these domains) are labeled in gold. Additional relevant DSL and DOS domain amino acids are labeled in red and blue, respectively, and further distinguished as being surface-exposed (white circles) or buried within the structure (green circles). A positively charged cluster of highly conserved surface-exposed residues within the DSL domain (labeled in red) identifies a putative Notch-binding surface (see text and Figure 3 legend). Interestingly, missense mutations associated with Teratology of Fallot in humans, and to autosomal dominant inner ear malformations in mice (i.e., headturner (Htu), slalom and Nodder) cluster near a common DOS region. Mutations in headturner and Teratology of Fallot affect amino acids buried under the surface defined by slalom and Nodder mutations and may impact the structure of the potential Notch binding site within DOS. Note that R252 (ALGS1) and Y255 (unique to Jagged; see Figure 3 legend) are also aligned with the putative Notch binding surfaces on the DSL and DOS domains. C–E) Modeling of specific substitutions of surface amino acids (highlighted in green) in the DOS domain results in realignment of the surface (e.g., Nodder and ALGS1; dashed white lines) or perturbations into space (e.g., slalom; dashed black line) that could potentially affect interactions with Notch. The exact topology of Notch/ligand interface remains to be explored by co-crystallization.