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Investigating the Molecular Details of the Intra- and Inter-domain Interactions within the Notch Regulatory Region:

The domain organization of the Notch receptor represents a highly conserved modular arrangement involving several sets of structural subunits.  The Notch extracellular domain (NEC) contains N-terminal EGF-like repeats followed by the negative regulatory region (NRR), which encompasses three tandemly repeated Lin-12 Notch modules (LNRs) and the HD domain.  The HD domain harbors a site for specific proteolytic cleavages  (S2), which S2 is crucial for the activation of the Notch receptor (Fig. 1). NEC is followed by the Notch transmembrane (NTM) domain and the intracellular Notch (ICN)

Figure 1.  Domain organization of the Notch Receptor and overview of Notch Signaling: The extracellular Notch is composed of an N-terminal ligand binding domain and the negative regulatory region (NRR) preceding the transmembrane regions of the protein. The NRR consists of three Lin12/Notch Repeats (LNRs) and the Heterodimerization domain (HD domain) which harbors the S1 and S2 cleavage sites. S3 cleavage site is on the intracellular side of the membrane and is followed by the intracellular cellular domain of Notch (ICN).  Ligand binding to the extracellular portion of Notch triggers metalloprotease cleavage at site S2. The resulting truncated transmembrane subunit of the receptor is a substrate for cleavage at S3 by γ-secretase, which releases the intracellular part of Notch (ICN) from the membrane. ICN migrates to the nucleus, where it assembles into a complex that turns on transcription of target genes. 

We had determined the X-ray crystal structures of human Notch1 and human Notch 2 NRRs (PDB ID#s: 1eto and 2oo4, respectively) during my postdoc years in Blacklow Laboratory at Harvard Medical School and determined them to be essentially identical.  The NRR is composed of three Lin12/Notch repeats (LNRs) and a Heterodimerization (HD) domain, which contains the S2 cleavage site. These are arranged in a cauliflower-like structure, in which the three LNR module ‘florets’ cover and protect the HD domain ‘stem’. (Fig. 2)

Figure 2: The crystal structure of human Notch2 NRR., LNR modules (red, blue, and green); HD domain yellow); arrows, positions of S1 (furin) and S2 (metalloprotease) cleavage.  The LNR modules wrap around the HD domain concealing the S2 site. 
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