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. Author manuscript; available in PMC: 2016 Jul 7.
Published in final edited form as: Structure. 2015 Jun 4;23(7):1227–1235. doi: 10.1016/j.str.2015.05.001

Insights into Autoregulation of Notch3 from Structural and Functional Studies of Its Negative Regulatory Region

Xiang Xu 1,2, Sung Hee Choi 1,2, Tiancen Hu 3, Kittichoat Tiyanont 1,2, Roger Habets 4, Arjan J Groot 4, Marc Vooijs 4, Jon C Aster 5, Rajiv Chopra 3, Christy Fryer 3, Stephen C Blacklow 1,2,5
PMCID: PMC4497832  NIHMSID: NIHMS691463  PMID: 26051713

Abstract

Notch receptors are transmembrane proteins that undergo activating proteolysis in response to ligand stimulation. A negative regulatory region (NRR) maintains receptor quiescence by preventing protease cleavage prior to ligand binding. We report here the X-ray structure of the NRR of autoinhibited human Notch3, and compare it with the Notch1 and Notch2 NRRs. The overall architecture of the autoinhibited conformation, in which three LNR modules wrap around a heterodimerization domain, is preserved in Notch3, but the autoinhibited conformation of the Notch3 NRR is less stable. The Notch3 NRR uses a highly conserved surface on the third LNR module to form a dimer in the crystal. Similar homotypic interfaces exist in Notch1 and Notch2. Together, these studies reveal distinguishing structural features associated with increased basal activity of Notch3, demonstrate increased ligand-independent signaling for disease-associated mutations that map to the Notch3 NRR, and identify a conserved dimerization interface present in multiple Notch receptors.

Graphical Abstract

graphic file with name nihms691463u1.jpg

The Notch signaling pathway influences numerous cell fate decisions during development and maintains tissue homeostasis in adults. Mammals have four Notch receptors and five canonical ligands, three homologous to Drosophila Delta (DLL1, DLL3, and DLL4), and two homologous to Drosophila Serrate (Jag1, Jag2). Whereas mammalian Notch1 and Notch2 are expressed in a wide variety of tissues and are essential for mammalian development, Notch3 expression is largely restricted to vascular smooth muscle. Thus, mice lacking Notch3 exhibit developmental defects limited primarily to vascular smooth muscle maturation and arterial specification (Domenga et al., 2004). Aberrant Notch3 expression has also been linked to a variety of different disease states, including the hereditary stroke syndrome CADASIL and ovarian cancer (Cancer Genome Atlas Research, 2011; Joutel et al., 1996; Park et al., 2006).

Normally, Notch receptors transmit signals by undergoing regulated proteolysis in response to transmembrane ligands presented on the surface of adjacent cells. The intrinsic resistance of Notch receptors to activating proteolysis is dependent on the integrity of a negative regulatory region (NRR), which encompasses a series of three LIN12-Notch repeats (LNRs) and a juxtamembrane “heterodimerization domain” (HD). The HD is cleaved during normal receptor maturation by a furin-like protease at a site called S1 (Logeat et al., 1998), but the NRR is resistant to further proteolysis in the absence of ligand (Gordon et al., 2007; Sanchez-Irizarry et al., 2004). Ligand stimulation induces receptor sensitivity to metalloprotease cleavage at a site called S2 (Brou et al., 2000; Groot et al., 2014; Mumm et al., 2000), which lies near the C-terminal end of the HD (Figure 1A). After metalloprotease cleavage, the truncated receptor, called NEXT, is primed for intramembrane cleavage at site S3 and additional sites by gamma secretase, which releases the intracellular part of Notch (NICD) from the membrane. NICD migrates to the nucleus, where it assembles a transcriptional activation complex that turns on the expression of Notch-responsive genes (Kopan and Ilagan, 2009).

Figure 1. Structure of the Notch3 NRR and comparison with NRRs from other Notch receptors.

Figure 1

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A. Top: domain organization of the Notch3 NRR. Below: Ribbon representation of the X-ray structure of the Notch3 NRR. The three LNR modules are colored in different shades of pink as in the schematic, and the HD domain is shaded cyan up to the furin cleavage site, and in darker blue C-terminal to the furin site. The three calcium ions are in green, disulfide bonds in yellow, and the sugar residues at the glycosylation site on the HD domain have a yellow carbon backbone with heteroatoms in CPK colors. The zoomed-in views shown in panels B and C are boxed in red. B–D. Comparison among Notch1 (gray; PDB ID code 3ETO), Notch2 (gold; PDB ID code 2OO4), and Notch3 (multicolored) NRRs. B. Zoomed in view of the LNR-A interface with LNR-B, highlighting the divergence of Notch3 at P1408, and its impact on the conserved cluster of tryptophan residues. The sugars have been removed for clarity. C. View focusing on the interface between the LNR-C module and the HD domain, illustrating the different packing arrangement in Notch3 compared to Notch1 and Notch2. D. Close-up view of the N-acetyl glucosamine residue attached to N1438 interacting with residues at the C-terminal end of helix 3 of the HD domain. See also Figure S1.

The X-ray structures of the Notch1 and Notch2 NRRs show that autoinhibition results from the masking of the metalloprotease cleavage site in the HD by the first two LNR modules and the linker between them (Gordon et al., 2009; Gordon et al., 2007). Moreover, mutations of the Notch1 NRR that are found in nearly half of human T cell acute lymphoblastic leukemia/lymphomas lead to increased ligand-independent signaling (Weng et al., 2004), highlighting the importance of the NRR in stabilizing the quiescent, “off” state of the receptor.

Here, we report the structure of the Notch3 NRR in its autoinhibited conformation. The structure reveals differences in packing interactions among the three NRRs that may account for the increased basal proteolytic sensitivity of Notch3. We also observe an increase in ligand-independent signaling associated with various point mutations that have been identified in different disease states, and identify a dimerization interface conserved among the Notch1, Notch2, and Notch3 receptors.

Results

Structure overview and unique features of the Notch3 NRR

The structure of the Notch3 NRR was solved to 2.4 Å resolution by X-ray crystallography, using the Notch 1 NRR (pdb ID 1ETO) as a search model for molecular replacement (Table 1; see methods). The Notch3 NRR adopts a compact structure, with the three LNR modules enveloping the HD domain in a closed, autoinhibited conformation (Figure 1A). The domain architecture is similar to the arrangement seen in the structures of the human Notch1 and Notch2 NRRs (Gordon et al., 2009; Gordon et al., 2007; Wu et al., 2010), and confirms that the structural basis for Notch3 autoinhibition results from the packing of the LNRs against the HD domain, precluding access of metalloprotease to the S2 cleavage site.

Table 1.

X-ray data collection and refinement statistics

Notch3 NRR
X-ray data collection
 X-ray source 24-ID-E (NECAT)
 Wavelength (Å) 0.9792
 Resolution (Å) 49.18 – 2.48 (2.57 – 2.48)
 Space group C 2
 Cell dimersions
  a, b, c (Å) 122.5, 64.3, 83.1
  α, β, γ (°) 90.0, 105.2, 90.0
 Rmerge* (%) 13.2 (78.6)
 CC(1/2) 99.2 (68.2)
 I/σ(I) 13.27 (2.23)
 Completeness (%) 99.99 (99.87)
 Redundancy 7.5 (7.5)
 Mosaicity 0.15
Model refinement
 Unique reflections 22,317 (2231)
 Subunits/asymmetric unit 2
 Rwork/Rfree 0.1829/0.2069 (0.2595/0.3157)
 Number of non-hydrogen atoms 3,701
  Macromolecules 3,450
  Ligand/ion(Ca2+) 96/6
  Water 155
 Protein residues 459
 Average B factor (Å2) 53.2
  Macromolecules 53.0
  Ligand/ion 78.3
  Water 41.3
 R.m.s deviations
  Bond lengths (Å) 0.004
  Bond angles (°) 0.83
 Ramachandran plot
  Favored (%) 97
  Allowed (%) 3
  Outliers (%) 0.22
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*

Rmerge = ΣhklΣi|Ii(hkl)i − <I(hkl)>|/ΣhklΣiIi(hk).

§

Numbers in parentheses refer to the highest resolution shell.

Detailed comparison of the Notch3 NRR to the Notch1 and Notch2 NRRs reveal substantial differences among the structures at the interdomain interfaces (Figure 1B,C). The backbone root mean square differences (RMSD) for superposition of the Notch3 NRR on that of Notch1 or Notch2 are 2.25 Å and 2.29 Å, respectively, as compared to a 1.65 Å RMSD for the Notch1/Notch2 NRR superposition (Figure S1). One important distinguishing feature of the Notch3 structure occurs at the interface between LNR-A and LNR-B (Figure 1B). Three absolutely conserved tryptophan residues (W1412, W1425, and W1434 in Notch3) play a critical role in stabilizing this interface. In Notch1 and Notch2, this aromatic cluster is further reinforced by an LNR-A histidine residue (H1471 in Notch1 and H1446 in Notch2), which engages the third tryptophan of the cluster in an aromatic π-stacking interaction. In Notch3, a proline residue (P1428) substitutes for histidine and makes only limited van der Waals contact with W1434, suggesting that this interface is less stable in the Notch3 NRR.

Another difference that distinguishes the Notch3 NRR is the nature of the interface between LNR-C and the first helix of the N-terminal part of HD domain (HD-N) (Figure 1C). In both Notch1 and Notch2, there is a salt bridge formed between an asparate in LNR-C and an arginine from the external face of the helix (D1534 to R1595 in Notch1, and D1506 to R1567 in Notch2). This linkage defines the interdomain orientation of the two modules and establishes the distance of closest approach between the helix and LNR-C. In Notch3, the analogous interaction becomes a hydrogen bond between a glutamate (E1472) on LNR-C and a glutamine (Q1533) on the helix. The shorter glutamine side chain at that position allows the helix to pack more tightly against LNR-C. This close-packed arrangement is further stabilized by an additional interaction between the same E1472 residue of LNR-C and R1534, an arginine residue at the next position of the helix.

An additional striking feature of the NRR from Notch3 is the position of the N-linked sugars attached to N1438 of LNR-B (Figure 1D). Both the first and second sugars of the chain exhibit well-defined electron density, assigned to two N-acetylglucosamine (GlcNAc) residues. Additional density is present for two more sugars, which are modeled as mannose (Figure S1C). The 1st GlcNAc forms several hydrogen bonds with nearby residues, including the side chain of R1619 and the backbone carbonyl oxygen of V1617. These two residues are located at the C-terminal end of helix 3 of the HD domain, and it is possible that these hydrogen bonding interactions between LNR-B and helix 3 help in stabilizing the closed conformation of the protein. Interestingly, both Notch1 and Notch2 have a consensus sugar modification site at the asparagine residue immediately preceding the first cysteine of LNR-B, which is positioned at the N-terminal end of helix 3. Although the Notch2 protein used to solve the X-ray structure was prepared in bacteria, and is thus unglycosylated, the Notch1 NRR solved in complex with an inhibitory Fab (PDB code: 3L95) was expressed in insect cells, and is glycosylated at this site (Wu et al., 2010). The sugar residues that are modeled in the structure also come within hydrogen bonding distance of helix 3, and may serve to increase the stability of the “off” state of these proteins as well.

The other main site of structural divergence among the three NRRs lies at the convergence point of the LNR-B and LNR-C modules with the HD domain (Figure S1D). In the Notch3 NRR, the connector between strand β4 and helix α3 containing the disulfide knuckle of the HD domain is not helical as in Notch1 and Notch2, but is instead an extended loop that projects farther from the core of the protein. The structural diversity in this region may also contribute to differential responsiveness of various receptors to different canonical ligands.

Structural basis for increased basal activity of Notch3

Our Notch3 NRR structure has two molecules in the asymmetric unit (ASU), with an RMSD of less than 1 Å between the two copies (Figure 2A). The most striking difference between the two copies resides in the LNR-A domain, which is partially disordered in one molecule (named mol-B), but is well defined in the other (named mol-A). The position of LNR-A is constrained by packing interactions with a symmetry related molecule in mol-A but not in mol-B, suggesting that LNR-A is more dynamic in the Notch3 NRR than anticipated. We revisited the question of LNR-A positional flexibility for the other Notch NRRs by calculating normalized B-factor values for the LNR-A domain relative to the rest of the structure for each Notch NRR in the PDB (Table S1). Although the absolute value of the B factor is affected by multiple influences including the quality of the dataset, the resolution, and crystal-packing interactions, the normalized B-factor ratio for the LNR-A domain of the Notch3 NRR is substantially higher than in other NRR structures (Table S1), consistent with the idea that the LNR-A module of Notch3 is more dynamic than its counterparts in Notch1 and Notch2.

Figure 2. Notch3 shows elevated basal activity.

Figure 2

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A. Superposition of the two copies of the Notch3 NRR in the asymmetric unit of the crystal. The copy labeled Mol-B shows disorder in the LNR-A module, shown in the close-up view (boxed). B, C. Notch3 shows higher amounts of reporter gene activity in the absence of ligand than either Notch1 or Notch2. Isogenic stable cell lines expressing Notch1, Notch2, or Notch3 chimeric receptors that report on a Gal4-response element (Malecki, 2006) were cultured either on plates coated with IgG control, or the DLL1 ligand (B), or in co-culture assays with OP9 stromal cells alone or stably expressing DLL1 (C). The firefly luciferase activity, normalized to a renilla luciferase control (see methods) was measured in cell lysates after 24 hours. In both B and C, assays were also performed in the presence of the gamma-secretase inhibitor compound E (GSI) as a control. Luciferase activity is normalized to the Notch1 signal with IgG control (B), or with OP9 control cells (C). Error bars represent the standard error of three replicates. Using a one-way ANOVA, statistically significant differences are indicated with asterisks: *** p < 10−3; **** p < 10−4. D. Urea dissociation assay. Conditioned media from 293T cells expressing either the Notch1 or Notch3 NRR were immunoprecipitated with anti-HA (α-HA)-coupled beads, followed by 30 min of incubation at room temperature in buffer with different concentrations of urea (0 to 4 M). Subunit dissociation was evaluated by SDS-PAGE followed by Western blot analysis with anti-FLAG and anti-HA antibodies, which are specific for the N- and C-terminal subunits, respectively. See also Figure S2.

We tested whether or not Notch3 exhibits a higher level of basal (ligand-independent) signaling activity than either Notch1 or Notch2 in U2OS cells using a reporter gene assay. These experiments were performed using isogenic stable cell lines with chimeric receptors (Gordon et al., 2009) that contain an intracellular Gal4-responsive element in order to eliminate potential confounding effects from endogenous receptors or differences in expression levels (Figure S2A, bottom schematic). We examined the responsiveness of the different receptors to the DLL1 ligand immobilized on the surface of the culture dish or on OP9 cells (Figure 2B,C), as well as to the Jag2 ligand expressed on NIH-3T3 cells (Figure S2B). In assays performed without ligand-expressing cells, with control cells that do not express the ligand, or with immobilized IgG in place of DLL1, the basal reporter activity of the Notch3-expressing cells is substantially higher than that seen in Notch1- or Notch2-expressing cells, even though the strength of the signal in the presence of either DLL1 or Jag2 is comparable for all three receptors (Figure 2B,C and Figure S2B). The amount of the mature Notch3 protein is comparable to that of Notch1 and less than that of Notch2 in the compared isogenic lines, and thus the increased basal activity of Notch3 cannot be attributed to an increased abundance of the protein (Figure S2C). Together, the increased level of ligand-independent Notch3 proteolysis and the increased positional disorder of the LNR-A domain in the Notch3 NRR crystal suggest that the autoinhibited conformation is more dynamic and/or less stable in Notch3 than in Notch1 or Notch2.

To determine experimentally whether the Notch3 NRR was less stable than the Notch1 NRR, we performed a subunit dissociation assay in which the integrity of the furin-processed NRR heterodimer was monitored as a function of added urea (Malecki et al., 2006). Whereas the subunits of the Notch1 NRR heterodimer remain non-covalently associated at concentrations up to 4 M urea as reported previously (Malecki et al., 2006), the Notch3 NRR is substantially more sensitive to urea-induced dissociation, indicating that the Notch3 NRR is less stable than the Notch1 NRR (Figure 2D).

Analysis of disease-associated mutations mapping to the Notch3 NRR

Mutations of Notch3 are responsible for the hereditary stroke syndrome known as CADASIL. Although the vast majority of the CADASIL mutations lie in the EGF-like repeats, there is one CADASIL mutation in the Notch3 heterodimerization domain, L1515P (Fouillade et al., 2008), which lies on the central beta strand of the HD domain (Figure 3A). In addition, a L1519P mutation has been reported to segregate with multiple affected individuals in a pedigree with familial autosomal-dominant infantile myofibromatosis (Martignetti et al., 2013), and an acquired S1580L mutation of Notch3 has been identified in the T cell acute lymphoblastic leukemia/lymphoma cell line TALL1, (Cancer Genome UK). L1519P is located in the linker region between β1 and β2 of the heterodimerization domain (Figure 3A), while S1580 is located in β-strand 4 of the hydrophobic core, with its side chain oxygen atom within hydrogen bonding distance of the backbone carbonyl oxygen atoms of L1519 and P1520.

Figure 3. Analysis of Notch3 disease-associated mutations.

Figure 3

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A. Cartoon close-up highlighting disease-associated sites of mutations and their interactions with nearby residues. B. Reporter gene assay examining the ligand-independent activity and Jag2-stimulated activity of full-length wild-type Notch3, and the S1580L, L1515P and L1519P mutated forms of Notch3. The firefly luciferase gene was under control of the TP1 promoter (Kurooka et al., 1998) and the Jag2 protein was expressed in stably transfected 3T3 cells (Luo et al., 1997). The ratio of firefly to control renilla luciferase activity is plotted both in the absence and presence of the gamma-secretase inhibitor compound E (GSI). Luciferase activity is normalized to the signal observed with vector alone co-cultured with 3T3 cells. Error bars represent the standard error of three replicates. Using a one-way ANOVA, statistically significant differences are indicated with asterisks: * p < 0.05; **** p < 10−4.

To test the effects of these mutations on ligand-independent and ligand-dependent signaling, we introduced each of these three mutations into the context of the full-length Notch3 protein (Figure S2A, top schematic), and tested the expression of a luciferase reporter gene under control of the Notch-responsive TP1 promoter in transient transfection assays (Kurooka et al., 1998). The reporter gene assays show that the S1580L mutation results in a large increase in ligand-independent signaling. The L1515P mutation, as previously reported, also produces a large increase in ligand-indpendent signaling (Fouillade et al., 2008), whereas L1519P results in a modest, but statistically significant increase in ligand-independent signaling (Figure 3B).

Dimerization of the Notch3 NRR

The Notch3 NRR crystal contains two copies of the protein in the asymmetric unit (ASU). The interface in the crystallographic dimer is entirely derived from surface residues on the LNR-C module (Figure 4A), which bury 1088 Å2 of surface area at the interface. The dimer interface present in the Notch3 NRR structure closely resembles the packing interface seen in all Notch NRR structures deposited in the protein data bank (Figures 4A and S3A), including two different X-ray structures of the isolated Notch1 NRR, a structure of a Notch1 NRR - Fab complex, and the structure of the isolated Notch2 NRR (PDB ID codes: 3ETO, 3I08, 3L95, and 2OO4 respectively). Multiple sequence alignment shows that this surface patch on LNR-C is highly conserved among all Notch NRRs (Figure 4B). Two key residues in this interface of the Notch3 NRR dimer are Y1474 and H1478, which form symmetric sandwich-like π-π interactions (Figure 4A, right panels). These reciprocal interactions are buttressed on each molecule by Y1471. Other important residues at the interface include R1483, D1485 and Q1486 from both molecules. Equilibrium sedimentation studies show that the Notch3 NRR does not conform to a single species model, and is best fit by a monomer-dimer equilibrium model with an estimated dimerization Kd of 30 μM (Figure S3B).

Figure 4. A conserved dimerization interface is observed in different NRR structures.

Figure 4

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A. Comparision of dimerization interfaces seen in the Notch3 NRR, the Notch1 NRR (pdb ID code 3ETO), and the Notch2 NRR (pdb ID code 2OO4). The left panels illustrate the mode of interaction between the two molecules, and the right panels, which represent the boxed regions, highlight conserved features among the three interfaces. B. Multiple sequence alignment showing conservation of the residues at the interface (left panel), and a surface representation of the structure (right panel) colored on a sliding scale from maroon to teal blue according the extent of sequence conservation using the program ConSurf (Ashkenazy et al., 2010). See also Figure S3.

To examine whether the LNR-C dimerization interface contributes functionally to Notch3 signaling, we created four mutated forms of the full-length Notch3 receptor designed to disrupt the dimer interface, Y1474E, Y1474R, H1478E, and H1478R, and tested the effect of these mutations on ligand-independent and ligand-dependent signaling in both plated ligand and cell co-culture assays. Both mutations of H1478 lead to significant increases in ligand-independent reporter gene activity compared with the unmutated receptor (Figures 5 and S4), suggesting that the residues at the dimerization interface play a role in maintaining the quiescent state of the receptor. Importantly, cells expressing the mutated receptors appear to reach the cell surface normally (Figure S4), and respond to ligand comparably to the wild-type receptor (Figure 5).

Figure 5. Effect of mutations of conserved residues in the dimer interface upon signaling.

Figure 5

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A. Reporter assay using plated ligand. Wild-type or mutated Notch3 receptors were cultured either on plates coated with IgG control or the DLL1 ligand, and treated with either DMSO carrier or a gamma secretase inhibitor. B. Reporter assay using co-cutured cells expressing the DLL1 ligand. Wild-type or mutated Notch3 receptors were co-cultured either with OP9 stromal cells alone or with OP9 cells stably expressing the DLL1 ligand, and treated with either DMSO carrier or a gamma secretase inhibitor. In both A and B, firefly luciferase activity, normalized to a renilla luciferase control, was measured in cell lysates after 24 hours. Luciferase activity is normalized to the signal observed for cells transfected with full-length Notch3 cultured on the plated IgG control (A), or with OP9 cells alone (B). Error bars represent the standard error of three replicates. Using a one-way ANOVA, statistically significant differences are indicated with asterisks: * p < 0.05; ** p < 0.01. See also Figure S4.

Discussion

Notch receptor quiescence is mediated by the NRR, which encompasses both the maturation cleavage site processed by a furin-like proprotein convertase and the metalloprotease cleavage site that undergoes the key activating cleavage in response to ligand stimulation. The structure and functional studies of the Notch3 NRR reported here deepen understanding of the architectural features shared among Notch NRRs that encode receptor autoinhibition, and highlight differences among the Notch1, Notch2, and Notch3 NRRs that distinguish their sensitivity to receptor activation.

Several lines of evidence support the conclusion that autoinhibition of the Notch3 NRR is less stringent than that of the NRR regions of Notch1 or Notch2. First, in the X-ray structure of the Notch3 NRR, the LNR-A domain has a higher average B-factor than the other domains of the protein (Table S1), and is more disordered than the analogous domain from Notch1 or Notch2 in the other NRR structures. In addition, the purified Notch3 NRR undergoes subunit dissociation at a much lower urea concentration, indicating that the Notch3 NRR is less stable than the Notch1 or Notch2 NRRs. Likewise, the reporter gene assay testing ligand-independent and ligand-dependent signaling shows that the basal, ligand-independent signal of Notch3 in the reporter assay using the Gal4 fusions with identical intracellular regions is significantly stronger than the basal signal from Notch1 or Notch2, even though the ligand-depdendent signal from all three receptors is comparable in the reporter assay. It remains unclear, however, why the basal activity of full-length Notch3 receptors in transient transfection assays is not as consistent, nor why gamma secretase inhibition (e.g. Figure 3B) does not consistently suppress basal activity of full-length receptors. One possibility is that the use of the Gal4 reporter system improves assay signal-to-noise by eliminating the confounding effects of endogenous receptor signaling. Another possibility is that the Notch3 intracellular domain, which is not part of the molecules used to examine basal activity (Figure 2), may contribute to restraint of basal signaling in the full-length protein, compensating in part for the reduced stability of the Notch3 NRR.

Increased basal Notch3 signaling activity might have particular relevance in pathogenic conditions where an increased amount of Notch3 is expressed. For example, NOTCH3 lies within a locus that is amplified in high-grade ovarian serous adenocarcinomas (Cancer Genome Atlas Research, 2011; Park et al., 2006), and Notch3 has also been implicated in the pathogenesis of certain lung cancers (Konishi et al., 2007).

Both loss-of-function and gain-of-function mutations of Notch3 receptors are also associated with human developmental disorders and cancer (Aster et al., 2011; Gridley, 2003; Joutel et al., 1996). The three mutations known to map to the heterodimerization domain of the Notch3 NRR include the CADASIL mutation L1515P, an infantile myofibromatosis mutation L1519P, and the S1580L mutation found in the TALL1 leukemia cell line. All three mutations tested here are associated with ligand-independent gain-of-function, suggesting that inhibitory antibodies capable of blocking ligand-independent signaling by binding the NRR might have therapeutic utility in these diseases.

Finally, the presence of a conserved dimerization interface among Notch1, Notch2, and Notch3, combined with the analytical ultracentrifugation data that fit best to a monomer-dimer equilibrium model, suggests that it possesses functional importance. The restricted distribution of molecules at the plasma membrane, the observation of receptor clustering in signaling complexes (Ahimou et al., 2004; Bardot et al., 2005; Nichols et al., 2007) and the presence of an additional weak dimerization interface within the intracellular ankyrin repeat domain of Notch (Allgood and Barrick, 2011; Nam et al., 2007; Nam et al., 2006) also point toward a potential role for dimerization in modulating signal transduction. Consistent with this idea, reporter gene assays suggest that autoinhibition is partially disrupted by mutations that destabilize the dimerization interface. However, a more detailed understanding of the role of cell-surface Notch dimers will require future study.

Methods

Protein expression and purification

For crystallization, the Notch3 NRR was subcloned into a modified pcDNA-series plasmid encoding the IgG kappa signal sequence, followed by the Notch3 NRR (1378–1640), and an in-frame gly-ser-his6 tag at the C-terminus. The encoded protein was expressed and secreted into the conditioned media by HEK293S GnTi− cells (Reeves et al., 2002). The media was collected after 4 days, and the NRR was bound to Ni-NTA beads (Qiagen) over a 3-hour incubation period at 4° C. After a wash with 50 mM HEPES buffer containing 500 mM NaCl and 20 mM imidazole, bound protein was eluted in 50 mM HEPES buffer containing 500 mM NaCl and 250 mM Imidazole. After buffer exchange into 50 mM tris, pH 8.0 containing 50 mM NaCl and 10 mM CaCl2, the Notch3 NRR protein was passed over a Hi-trap Q HP column and eluted using an NaCl gradient. The recovered Notch3 NRR was incompletely processed into two subunits by furin, as judged by SDS-PAGE. Thus, the protein was treated with furin (30 U/mg) at room temperature for 6 hours, and the fully processed protein was further purified by size exclusion chromatography on a Superdex 75 HR 10/30 column after buffer exchange into 20 mM HEPES buffer, pH 7.4, containing 150 mM NaCl and 10 mM CaCl2. The protein was concentrated to 45 mg/mL for initial crystallization screening.

Crystallization, Data collection, and Structural refinement

Crystallization trials were carried out using the sitting drop vapor diffusion method at both 22° C and 4° C. Initial crystals were obtained in 2 conditions: one at 4° C containing 0.2 M ammonium sulfate, 15% PEG4000, and 0.1M tri-sodium citrate, pH 5.6, and the other at 22° C containing 20% isopropanol, 20% PEG4 000, and 0.1 M tri-sodium citrate, pH 5.6. After iterative refinement, final crystals were grown at 22° C by mixing equal volumes of Notch3 NRR at 5 mg/ml with a precipitant solution containing 0.2 M sodium acetate, 20% PEG4000, and 0.1 M tri-sodium citrate at pH 5.4. Plate-like crystals typically grew within 2 weeks reaching a maximal size of 0.05×0.5×0.5 mm3. For data collection, a single crystal was flash frozen from a precipitant solution containing 20% (v/v) glycerol.

Diffraction data were collected from a flash-cooled crystal at 100°K using the 24ID-E beam line at Argonne National Laboratory and were processed, integrated, and scaled together with HKL2000 (Otwinowski and Minor, 1997). The criterion used for data inclusion was based on the value of CC1/2 (Karplus and Diederichs, 2012). The Notch3 NRR structure was solved by molecular replacement (MR) as implemented in Phenix (Adams et al., 2010) using a Notch1 NRR structure (PDB ID code 3ETO) as the search model. Structure refinement was carried out using conjugate-gradient energy minimization, torsion-constrained molecular dynamics simulated annealing, group B factor refinement, individual B factor refinement, and TLS protocols (Translation/Libration/Screw) in Phenix with 5% of the reflections omitted for free R factor calculation. Electron density peaks in difference Fourier maps at a height of above 3σ were assigned as water molecules in later refinement stages if they had reasonable geometry in relation to hydrogen bond donors or acceptors and their B-factors did not rise above 50 Å2 during subsequent refinement. Model building was performed in Coot (Emsley and Cowtan, 2004) guided by σA-weighted 2Fo-Fc maps, Fo-Fc maps, and composite omit maps. All structure figures were generated using Pymol v1.7 (Schrodinger, 2010).

Luciferase reporter assay

Luciferase reporter gene assays were carried out using methods previously described (Gordon et al., 2009). For the studies in Figure 2 and S2B, experiments were performed using isogenic stable cell lines with chimeric Notch1-, Notch2-, or Notch3-Gal4 fusions, and the reporter was a Gal4-responsive element. For studies testing disease associated or dimer interface mutations, the full-length Notch3 protein was used in transient transfections of U2OS cells, and the reporter was the TP1 Notch-responsive element. In plated ligand assays, the various Notch-expressing cells were cultured on tissue culture plates treated with either soluble DLL1 ligand (spanning from the N-terminus through EGF-like repeat 5 (Andrawes et al., 2013)) or an IgG control protein in the absence or presence of a gamma secretase inhibitor, compound E (1μM). In the co-culture assays, OP9 cells alone or stably expressing DLL1, or 3T3 cells alone or stably expressing Jag2 were used as signal sending cells, again in the absence or presence of a gamma secretase inhibitor, compound E (1μM).

Urea dissociation assay

Urea dissociation assays were performed essentially as described previously (Malecki et al., 2006). The Notch1 (Malecki et al., 2006) or Notch3 NRR (S1382–S1640, flanked by N-terminal FLAG and C-terminal HA tags) was recovered from conditioned media using anti-HA antibody-conjugated beads in 50mM Tris buffer, pH7.5, containing 150mM NaCl and 0.1% NP40, and subjected to varying concentrations of urea for a period of 30 minutes before recovery of the beads by centrifugation. After three rapid washes in the incubation buffer, the proteins retained on the beads were analyzed by Western blotting.

Analytical ultracentrifugation

Analytical ultracentrifugation was performed in six-sectored cells using a Beckman Coulter Optima XL-A analytical ultracentrifuges equipped with an An-60 Ti analytical rotor. Before centrifugation, wild-type Notch3 NRR protein was extensively dialyzed into 25mM HEPES buffer, pH7.5, containing 150 mM NaCl, and 5mM CaCl2. Protein concentrations of 3, 9, and 27 μM were prepared by dilution of a stock solution with the dialysate, and the dialysate was used in the three reference channels. Samples were centrifuged at 14,000 and 18,000 rpm at 4°C until equilibrium was reached, as judged by the stability of absorption scans taken at 230 nm, 280 nm and 290 nm wavelengths over two consecutive 2-hour intervals. Data for final analysis were acquired by averaging 10 scans at each wavelength using 1 nm steps, and only data in the linear range of the instrument (between 0.01–1 AU) were used for subsequent analysis. Global fitting was carried out using the Origin 6.0 software package comparing fits to distinguish between a single-species model and a monomer-dimer equilibrium, and to estimate a value for the dimerization Kd. Plots comparing fits to a monomer-dimer equilibrium with a 30 μM value of Kd (left panel) or a single species monomer (right panel) are shown for 280 nm scan of the 9 μM sample spun at 18,000 rpm in Figure S3B.

Immunofluorescence and flow cytometry

Detection of various Notch proteins on the surface of U2OS cells was carried out using a mouse IgG anti-Notch3 antibody A13 (Li et al., 2008), followed by secondary detection with goal anti-mouse IgG coupled to Alexa546. Flow cytometry was carried out using the same antibodies, using a BD Accuri™ C6 instrument. The data were analyzed using BD Accuri™ C6 software.

Supplementary Material

1
2

Highlights.

  • Structure of the human Notch3 negative regulatory region in its autoinhibited state

  • Low intrinsic stability of autoinhibited Notch3 results in detectable basal activity

  • Structure reveals a conserved dimerization interface among Notch NRRs

Acknowledgments

This work was supported by NIH grant R01 CA092433 (to SCB), and P01 CA119070 (to JCA and SCB). MV, AJ and RH are supported by European Research Council ERC grants 208259 and 334987.

Footnotes

Accession codes

Coordinates of the Notch3 NRR have been deposited in the PDB with accession code 4ZLP.

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