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First published online September 17, 2008
doi: 10.1242/10.1242/jcs.035683

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The molecular logic of Notch signaling – a structural and biochemical perspective

Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA

View larger version (81K): [in this window] [in a new window]   Fig. 1. Notch signaling pathway, and domain organization of Notch receptors and DSL ligands. (A) Model for the major events in the Notch signaling pathway. Signals initiated by the engagement of ligand (1) lead to metalloprotease (MP) cleavage at site S2 (2). This proteolytic step allows the cleavage of Notch by the -secretase complex at site S3 within the transmembrane domain (3) and release of intracellular notch (ICN) from the membrane. ICN translocates to the nucleus (4), where it enters into a transcriptional activation complex with CSL and MAM, inducing the transcription of target genes (5). (B,C) The domain organization of Notch receptors (B) and DSL-family ligands (C) from fly, human and worm. TM, transmembrane domain; NEC, Notch extracellular subunit.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Structures of ectodomain fragments of both NOTCH1 and JAGGED1, and models for the Notch ectodomain. (A) NMR structure of EGF-like repeats 11-13 from human NOTCH1 (PDB ID code 1TOZ). Two of the 20 calculated structures in the ensemble are shown: the double-headed arrow indicates the range of positions that are occupied by repeat 13 among the 20 calculated structures. Disulfide bonds are colored orange; hydrophobic residues engaged in interdomain contacts are shown as sticks. Bound Ca2+ ions, positioned on the basis of structural alignment with other Ca2+-binding EGF-like repeats, are shown as green spheres. (B,C) Two proposed models for the overall organization of a Notch ectodomain. In B, a rod-like, extended model is shown, whereas C illustrates one possible compact model. EGF-like repeats (ovals) are shaded dark purple when they contain a consensus Ca2+-binding site (per the definition used by UniProt) and light purple when they do not. (D) Representation of the X-ray structure for the JAGGED1 polypeptide (PDB code 2VJ2), comprising the DSL domain and EGF-like repeats 1-3. Disulfide bonds are yellow. Side-chains of residues at the proposed Notch-binding surface are rendered as colored sticks.

View larger version (102K): [in this window] [in a new window]   Fig. 3. X-ray structure of the human NOTCH2 NRR in the autoinhibited conformation and models for signal activation. (A) Ribbon representation of the NOTCH2 NRR. The LNR modules are colored different shades of pink and purple, and the HD domain is colored in white and turquoise; the white and turquoise represent residues that are N- and C-terminal, respectively, to the furin-cleavage loop (S1). The three bound Ca2+ ions are green, the bound Zn2+ ion is purple and the ten disulfide bonds are red. The positions of S1 and S2 cleavage are indicated with red arrows. (B) The LNR-AB linker sterically blocks access to the metalloprotease-cleavage site. The hydrophobic pocket in the HD domain that houses the S2 site is rendered in a surface representation, and residues from the LNR-AB linker are in ball-and-stick representation. (C) Model for activation by mechanical force. Endocytosis of bound ligand generates a mechanical force that tugs on the LNR domain (pink structure; 1), disengaging the hydrophobic plug from the hydrophobic pocket containing the S2 site (2) and peeling the LNR repeats away from the HD domain (white structure; 3). Partial or complete relaxation of the HD domain then allows access of the metalloprotease to the S2 site and cleavage of the scissile bond to trigger Notch activation (4). (D) Peeling of the LNR repeats (pink) away from the HD domain (white) might not confer sufficient exposure of the S2 site to allow cleavage by metalloprotease. The left panel depicts a hypothetical model for the negative regulatory region upon peeling of the first two LNR repeats away from the HD domain; the right panel shows the structure of the catalytic domain of the metalloprotease TACE (PDB ID code 1BKC). The deep active-site cleft is indicated. A and B are adapted from Gordon et al. (Gordon et al., 2007) and are reproduced with permission.

View larger version (139K): [in this window] [in a new window]   Fig. 4. Ribbon diagrams representing the structure of human and worm Notch ternary complexes. (A, left) Human complex of the ANK domain of NOTCH1 (blue), CSL (green) and the N-terminal region of MAML1 (red) bound to an 18 base-pair DNA sequence from the hes1 promoter (PDB code 2F8X). (A, right) Worm complex of the RAMANK region of LIN-12 (ANK and RAM are blue and cyan, respectively), LAG-1 (green), and the N-terminal region of SEL-8 (red) (PDB code 2FO1). The structures illustrate the cooperative binding of MAM to a composite surface that is created at the interface between the Notch ANK domain and CSL. Bottom panels show a 105° rotation around the vertical axis. (B) Superposition of CSL structures, showing the difference in loop conformation between CSL-DNA complexes and complexes that include RAM domains, ANK domains or both. Mouse CSL-DNA (PDB code 3BRG, magenta) and worm CSL-DNA (PDB code 1TTU, green) structures have a `closed' loop. Worm RAM-CSL-DNA (PDB code 3BRF, yellow; PDB code 3BRD, cyan), human ANK-MAM-CSL-DNA (red) and worm RAMANK-MAM-CSL-DNA (blue) complexes have an `open' loop. (C) Superposition of worm (bright) and human (faded) ternary-complex structures. Several unique insertions at the N-terminus of RAM and within the ANK domain are found in worm LIN-12 but not in other Notch molecules (orange). These features might play a role in the more compact packing of the worm NTC structure when compared with the human NTC structure.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Ribbon diagram of two symmetry-related copies of the human NTC (PDB code 2F8X), revealing the near-linear orientation of the two DNA elements, which mimics the inverted repeat of the SPS element. Superposition of the symmetry-related pseudo-dimer (DNA in yellow, with CSL-binding sites in orange) on ideal B-form DNA corresponding to 42 base pairs of the hes1 promoter (gray, with CSL-binding sites in black) reveals that the orientation and spacing between the two CSL sites in the crystal approximates, but does not match, the expected spacing and orientation in a natural SPS.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Model for the assembly of Notch ternary complexes. A high-affinity interaction between the N-terminal RAM peptide of Notch and the BTD of CSL is likely to be the first event in the assembly of Notch transcriptional activation complexes. This step allows the lower-affinity ANK domain to bind at its docking site, resulting in ordering of the ankyrin-like N-cap and first repeat of the ANK domain. The interface between the ANK domain and the RHR-N (N) and RHR-C (C) domains of CSL create a composite surface for the binding of MAM, which recruits p300 or CBP. Higher-order homotypic assemblies of Notch complexes or heterotypic assemblies with other transcription factors might be required for the transcription of specific Notch targets.

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