The negative regulatory region (NRR) of the Notch receptor, which is sandwiched between the ligand binding and transmembrane domains, harbors the structural machinery necessary to maintain metalloprotease resistance in the absence of ligand. The NRR contains three cysteine-rich Lin12/Notch repeats (LNRs) and a “heterodimerization domain” (HD) that contains both the S1 and S2 cleavage sites. Several lines of evidence accumulated over many years led to the conclusion that the NRR is the “activation switch” of the receptor. Receptors that lack the EGF-like repeats are functionally inert (Kopan et al., 1996
; Lieber et al., 1993
; Rebay et al., 1993
; Struhl and Adachi, 1998
). By contrast, deletion of the LNR modules or point mutations of key residues within them lead to gain-of-function phenotypes (Greenwald and Seydoux, 1990
) and ligand-independent metalloprotease cleavage (Sanchez-Irizarry et al., 2004
). The regulatory importance of the NRR is also highlighted by the fact that most Notch1 point mutations and insertions that are found in patients who have T-cell acute lymphocytic leukemia (T-ALL) are located within the NRR (Weng et al., 2004
In the crystal structure of the NRR from human NOTCH2, the protein adopts an autoinhibited conformation in which the LNR and HD domains interact extensively to bury the metalloprotease site (Gordon et al., 2007
). The LNR domain covers the HD domain, much like a mushroom cap protecting its stem (). Similar to the prototype LNR from human NOTCH1 that was solved by NMR (Vardar et al., 2003
), each LNR module has an irregular fold with little secondary structure and is held together by three disulfide bonds and a calcium ion, coordinated by several acidic residues. The two subunits of the HD domain are intimately intertwined in an α-β sandwich, and constitute a single protein domain that resembles the SEA domains of mucins (Maeda et al., 2004
). The poorly conserved loop that encompasses the S1 cleavage site, which was excised to facilitate crystallization, is distant from the metalloprotease-cleavage site S2.
Figure 3 X-ray structure of the human NOTCH2 NRR in the autoinhibited conformation and models for signal activation. (A) Ribbon representation of the NRR. The LNR modules are colored different shades of pink and purple and the HD domain is colored in two shades (more ...)
The interface between the LNR and HD domains buries 3000 Å2 of surface area and the molecular details of these interactions provide the structural basis for the metalloprotease resistance of Notch in the absence of ligand. Three highly conserved residues that are derived from the linker that connects LNRs A and B “plug” the small hydrophobic pocket that contains the metalloprotease site, thereby sterically occluding it (). The preceding helix from the HD domain, which is anchored over the S2 site by hydrophobic interactions with elements from the LNR domain, also precludes metalloprotease access. The structure also suggests that the LNR domain imparts global stabilization to the NRR via its extensive interactions with the HD domain and, most significantly, provides direct evidence that the LNR repeats must be displaced to unmask the S2 site of the HD and allow metalloprotease cleavage.
A key question that remains is how are the LNRs displaced to expose the S2 site? One model, first suggested by Muskavitch to rationalize trans
-endocytosis of the Notch ectodomain into ligand-expressing cells, is that bound ligand induces mechanical strain on the Notch receptor (Parks et al., 2000
). In light of the NRR structure, this process can be envisioned as one in which ligand stimulation exerts a mechanical force on the Notch receptor, pulling the LNR repeats away from the HD domain to expose the S2 site (). In a mechanotransduction “lift and cut” model with the NRR as the mechanosensor, endocytosis would then be the source of the mechanical force that is needed to peel the protective LNR modules away from the HD domain. This idea is consistent with the known requirement for endocytosis of ligands in Notch signal transduction, and with the finding that soluble ligands typically do not activate Notch receptors (Sun and Artavanis-Tsakonas, 1997
). The alternative model for activation would be an allosteric model, in which ligand engagement trips an allosteric switch that disengages the LNR modules from the HD domain. Clearly, additional studies are needed to distinguish between these two models for activation.
In either an allosteric or mechanotransduction model for Notch activation, it is unlikely that the activating metalloproteases can gain access to the Notch S2 site after mere stripping of the LNR modules away from the HD domain, because the active sites of metalloproteases such as tumor-necrosis factor-α (TNF-α)-converting enzyme (TACE; also known as ADAM17) lie in a deep cleft (). Thus, we would expect that certain key secondary structural elements of the HD domain will also unravel after displacement of the LNR repeats, generating an “open” conformation that renders the S2 site accessible to the protease. Such conformational changes in the HD domain might include localized movement or melting of helix3, anchored above the cleavage site (), to release the strand that contains the S2 site, or even complete unfolding of the HD domain with accompanying subunit dissociation (Nichols et al., 2007
The discovery that mutations are frequently found in the HD domain of NOTCH1 in human T-ALL moved NOTCH1 to the forefront in understanding disease pathogenesis and also pointed to the NOTCH1 NRR as a mechanism-based therapeutic target. These mutations, which map primarily to the highly conserved hydrophobic interior of the HD, lead to ligand-independent increases in signaling, suggesting that domain destabilization facilitates ligand-independent S2 cleavage and subsequent receptor activation (Malecki et al., 2006
; Weng et al., 2004
). Recently, inhibitory and activating antibodies against the NRR from human NOTCH3 were reported (Li et al., 2008
); the epitope of the inhibitory antibody includes residues from both the LNR-A and HD domains, consistent with the notion that it clamps the NRR in its metalloprotease-resistant conformation. Importantly, the existence of modulatory antibodies constitutes proof of principle, showing that it is possible to identify mechanism-based therapeutics that turn Notch signaling on or off via the NRR. Additional studies to define the structural characteristics of the “on” state of the NRR in normal and disease-associated signaling should also help to guide the development of mechanism-based modulators of signaling.