Notch1 Negative Regulatory Region Research Articles

Secretory expression of negative regulatory region of human Notch1 in Escherichia coli and preparation of a functional polyclonal antibody.

Notch signaling is a highly conserved pathway existed in multicellular organisms. It plays roles in normal human body development, human cancer initiation, progression and metastasis. The Notch negative regulatory region (NRR) is critical for Notch signaling, and cleavage at the S2 site in the NRR ultimately leads to the activation of Notch signaling. To study the function of human NRR1, we expressed the recombinant human NRR1 (rhNRR1) domain in Escherichia coli. After purification, rhNRR1 was obtained with approximately 94% purity according to SDS-PAGE analysis. Furthermore, the polyclonal anti-rhNRR1 serum raised by immunizing mouse with the purified rhNRR1 was able to reduce the generation of active form of Notch1 intracellular domain in HeLa cells, which implied the raised antibody could recognize and bind the natural conformation of Notch1 NRR. Preparation of rhNRR1 by this way is convenient, time-consuming, and could be used to the preparation of anti-NRR1 therapeutic antibody.

Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia.

Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML.

Human NOTCH2 Is Resistant to Ligand-independent Activation by Metalloprotease Adam17

Cell surface receptors of the NOTCH family of proteins are activated by ligand induced intramembrane proteolysis. Unfolding of the extracellular negative regulatory region (NRR), enabling successive proteolysis by the enzymes Adam10 and γ-secretase, is rate-limiting in NOTCH activation. Mutations in the NOTCH1 NRR are associated with ligand-independent activation and frequently found in human T-cell malignancies. In mammals four NOTCH receptors and five Delta/Jagged ligands exist, but mutations in the NRR are only rarely reported for receptors other than NOTCH1. Using biochemical and functional assays, we compared the molecular mechanisms of ligand-independent signaling in NOTCH1 and the highly related NOTCH2 receptor. Both murine Notch1 and Notch2 require the metalloprotease protease Adam17, but not Adam10 during ligand-independent activation. Interestingly, the human NOTCH2 receptor is resistant to ligand-independent activation compared with its human homologs or murine orthologs. Taken together, our data reveal subtle but functionally important differences for the NRR among NOTCH paralogs and homologs.

Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability

The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force – the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain – both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.

Identification of novel NOTCH1 mutations: Increasing our knowledge of the NOTCH signaling pathway

Alterations in the NOTCH1 signaling pathway are found in about 60% of pediatric T-ALL, but its impact on prognosis remains unclear. We extended the previously published CoALL cohort (n = 74) to a larger cohort (n = 127) and additionally included 38 Argentine patients from ALL IC-BFM to potentially identify novel mutations and decipher a stronger discriminatory effect on the genotype/phenotype relationship with regard to early treatment response and long-term outcome. Overall, 101 out of 165 (61.2%) T-ALL samples revealed at least one NOTCH1 mutation, 28 of whom had combined NOTCH1 and FBXW7 mutations. Eight T-ALL samples (4.8%) exclusively revealed FBXW7 mutations. Fifty-six T-ALL (33.9%) exhibited a wild-type configuration of either gene. Four novel NOTCH1 mutations were identified localized in the C-terminal PEST domain, in the rarely affected LNR repeat domain and in the ankyrin domain. Novel LNR mutations may contribute to a better understanding of the structure of the NOTCH1 negative regulatory region (NRR) and the R1946 mutation in the ankyrin domain may represent an unusual loss-of-function mutation. Overall, NOTCH1 pathway mutations did not affect the relapse rate and outcome of the extended T-ALL cohort uniformly treated according to CoALL protocols, although NOTCH1 mutations were associated with good response to induction therapy (P = 0.009). Individually, HD and PEST domain mutations might exert distinct functional effects on cellular homeostasis under treatment NOTCH1 pathway activity with prognostic implications.

Insights into Notch3 Activation and Inhibition Mediated by Antibodies Directed against Its Negative Regulatory Region

Notch receptors are single-pass transmembrane proteins that regulate development and tissue homeostasis in all metazoan organisms. Prior to ligand-induced signaling, Notch receptors adopt a proteolytic resistant conformation maintained by a critical interdomain interface within a negative regulatory region (NRR), which sits immediately external to the plasma membrane. Signaling is initiated when ligand binding induces exposure of the proteolytic cleavage site, termed S2, within the NRR. Here, we use hydrogen exchange in conjunction with mass spectrometry to study the dynamics of the human Notch3 NRR in four distinct biochemical states: in its unmodified quiescent form, in a proteolytically “on” state induced by ethylenediaminetetraacetic acid, and in complex with either agonist or inhibitory antibodies. Induction of the on state by either ethylenediaminetetraacetic acid or the agonist monoclonal antibody leads to accelerated deuteration in the region of the S2 cleavage site, reflecting an increase in S2 dynamics. In contrast, complexation of the Notch3 NRR with an inhibitory antibody retards deuteration not only across its discontinuous binding epitope but also around the S2 site, stabilizing the NRR in its “off” state. Together with previous work investigating the dynamics of the Notch1 NRR, these studies show that key features of autoinhibition and activation are shared among different Notch receptors and provide additional insights into mechanisms of Notch activation and inhibition by modulatory antibodies.

Abstract 231: Conformation-specific monoclonal antibodies against Notch1 target aberrant Notch1 signaling in cancer stem cells.

Abstract Notch signaling pathway plays a key role in regulating various cell fates including those of stem cells, throughout the mammalian development and adult homeostasis. Here we report characterization of anti-Notch1 monoclonal antibodies (mAb's) with potential therapeutic applications. Antibodies against various domains of human-Notch1 receptor were generated and characterized, mAb's generated against primary ligand-binding domain (EGF like repeats 11-12) and activation domain NRR (Negative Regulatory region) showed most prominent therapeutic potential. The Notch1 specific mAb 602.101 against ligand-binding domain (EGF like repeats 11-12) prevents binding of Jagged and Delta-like ligands and subsequent Notch1 receptor activation in breast cancer stem cells. Further, antibody treatment leads to apoptosis, inhibition of epithelial to mesenchymal transitions as well as irreversible depletion of cancer stem cells. The conformation specific mAb's against Notch1 NRR can specifically distinguish between the mutant and the wild type human Notch1 receptor present on cancer cells. Anti-NRR mAb's 604.107 (mutant specific) and 604.132 (wild type specific) specifically inhibited Notch1 signaling in leukemic and breast cancer stem cells. These mAbs can serve as a potential tool to irreversibly combat therapy resistant cancer stem cell subpopulations and to inhibit the epithelial to mesenchymal transitions in various types of cancers. Citation Format: Ankur Sharma, Annapoorni Rangarajan, Rajan R. Dighe. Conformation-specific monoclonal antibodies against Notch1 target aberrant Notch1 signaling in cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 231. doi:10.1158/1538-7445.AM2013-231

A Forced Opening

Biochemistry![Figure][1] CREDIT: N. L. STEPHENSON AND J. M. AVIS, PNAS ADVANCED ONLINE EDITION, 24 SEPTEMBER 2012, REPRODUCED WITH PERMISSION FROM PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES U.S.A. Notch proteins are transmembrane receptors that have major roles in the regulation of cell proliferation and development, and mutations in the Notch signaling pathway contribute to human diseases and cancer. When Notch proteins are bound by their ligands, which are themselves transmembrane proteins found on adjacent cells, endocytosis of the Notch extracellular domain into the ligand-bearing cell occurs, during which the Notch protein is cleaved, allowing the intracellular domain to be translocated to the nucleus, where it activates target genes. Stephenson and Avis used atomic force microscopy (AFM) to physically stretch a portion of the receptor called the Notch negative regulatory region, which contains the masked site of proteolysis. They could show through AFM measurements and molecular dynamics simulations that such a mechanical stimulus unfolded the protein to expose a site where protolytic cleavage occurred (and actually monitored the latter as release of the AFM tip). The events and forces measured were consistent with a favored model of Notch signaling in which forces of ligand endocytosis expose the otherwise buried cleavage site, leading to slicing of the receptor and initiation of signaling. Proc. Natl. Acad. Sci. U.S.A. 109 , 10.1073/pnas.1205788109 (2012). [1]: pending:yes

Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region

The conserved Notch signaling pathway plays crucial roles in developing and self-renewing tissues. Notch is activated upon ligand-induced conformation change of the Notch negative regulatory region (NRR) unmasking a key proteolytic site (S2) and facilitating downstream events. Thus far, the molecular mechanism of this signal activation is not defined. However, strong indirect evidence favors a model whereby transendocytosis of the Notch extracellular domain, in tight association with ligand into the ligand-bearing cell, exerts a force on the NRR to drive the required structure change. Here, we demonstrate that force applied to the human Notch2 NRR can indeed expose the S2 site and, crucially, allow cleavage by the metalloprotease TACE (TNF-alpha-converting enzyme). Molecular insight into this process is achieved using atomic force microscopy and molecular dynamics simulations on the human Notch2 NRR. The data show near-sequential unfolding of its constituent LNR (Lin12-Notch repeat) and HD (heterodimerization) domains, at forces similar to those observed for other protein domains with a load-bearing role. Exposure of the S2 site is the first force "barrier" on the unfolding pathway, occurring prior to unfolding of any domain, and achieved via removal of the LNRAB linker region from the HD domain. Metal ions increase the resistance of the Notch2 NRR to forced unfolding, their removal clearly facilitating unfolding at lower forces. The results provide direct demonstration of force-mediated exposure and cleavage of the Notch S2 site and thus firmly establish the feasibility of a mechanotransduction mechanism for ligand-induced Notch activation.

NOTCH1 Signaling Defines a Leukemia Initiating Cell Population in T-Cell Acute Lymphoblastic Leukemia

Abstract 1507Leukemia initiating cells (LIC) contribute to therapeutic resistance as a result of their capacity to accumulate mutations in pathways, such as the NOTCH1 receptor signaling pathway, that promote self-renewal and survival within specific niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in driving therapeutic resistance. However, the role of NOTCH1 activation in human T-ALL LIC propagation and LIC sensitivity to selective NOTCH1 receptor inhibition has not been examined. The difficulties in maintaining primary cultures of leukemia cells have hampered investigations into the biology of T-ALL LIC and underscore the need for a direct transplantation model to characterize human LIC in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL LIC. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2Rƒn gamma null (NSG) mice, was identified in primary adult T-ALL without an in vitro expansion. In this study, we identified and molecularly characterized potential LIC populations in pediatric T-ALL without preceding in vitro culture and examined the role of NOTCH1 activation in LIC propagation. To further define the T-ALL LIC, CD34+CD2+CD7+ or CD34+CD2+CD7− cells were isolated from T-ALL primary patients’ blood by FACS sorting and transplanted into neonatal RAG2−/− gamma chain−/− mice to determine their leukemic engraftment potential. Limiting dilution experiments were performed with cells from six T-ALL patient samples. Mice transplanted with CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale bone marrow and enlarged spleen, thymus and liver. Hematopoietic organs were analyzed by flow cytometry and showed engraftment of bone marrow, spleen, thymus and liver. Furthermore, the disease could be serially transplanted. LIC were uniquely susceptible to targeted inhibition in vivo with a therapeutic human NOTCH1 negative regulatory region selective monoclonal antibody (mAb) while normal human hematopoietic progenitors were spared thereby highlighting the cell type and context specific effects of NOTCH signaling. Both the NOTCH1 mAb treatment and lentiviral shRNA knockdown of NOTCH1 reduced NOTCH1, HES1 and c-MYC transcript levels, underscoring the selectivity of NOTCH1 mAb inhibition of NOTCH signaling. These results demonstrate that CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL. Moreover, inhibition of NOTCH signaling by either mAb or shRNA-mediated Notch1 knockdown might be another strategy to target the LIC in T-ALL. Disclosures:No relevant conflicts of interest to declare.

Force-induced unfolding simulations of the human Notch1 negative regulatory region: possible roles of the heterodimerization domain in mechanosensing.

Notch receptors are core components of the Notch signaling pathway and play a central role in cell fate decisions during development as well as tissue homeostasis. Upon ligand binding, Notch is sequentially cleaved at the S2 site by an ADAM protease and at the S3 site by the γ-secretase complex. Recent X-ray structures of the negative regulatory region (NRR) of the Notch receptor reveal an auto-inhibited fold where three protective Lin12/Notch repeats (LNR) of the NRR shield the S2 cleavage site housed in the heterodimerization (HD) domain. One of the models explaining how ligand binding drives the NRR conformation from a protease-resistant state to a protease-sensitive one invokes a mechanical force exerted on the NRR upon ligand endocytosis. Here, we combined physics-based atomistic simulations and topology-based coarse-grained modeling to investigate the intrinsic and force-induced folding and unfolding mechanisms of the human Notch1 NRR. The simulations support that external force applied to the termini of the NRR disengages the LNR modules from the heterodimerization (HD) domain in a well-defined, largely sequential manner. Importantly, the mechanical force can further drive local unfolding of the HD domain in a functionally relevant fashion that would provide full proteolytic access to the S2 site prior to heterodimer disassociation. We further analyzed local structural features, intrinsic folding free energy surfaces, and correlated motions of the HD domain. The results are consistent with a model in which the HD domain possesses inherent mechanosensing characteristics that could be utilized during Notch activation. This potential role of the HD domain in ligand-dependent Notch activation may have implications for understanding normal and aberrant Notch signaling.

Evidence for Increased Exposure of the Notch1 Metalloprotease Cleavage Site upon Conversion to an Activated Conformation

Notch proteins are transmembrane receptors that normally adopt a resting state poised to undergo activating proteolysis upon ligand engagement. Receptor quiescence is maintained by three LIN12/Notch repeats (LNRs), which wrap around a heterodimerization domain (HD) divided by furin cleavage at site S1 during maturation. Ligand binding initiates signaling by inducing sensitivity of the HD to proteolysis at the regulated S2 cleavage site. Here, we used hydrogen exchange mass spectrometry to examine the solution dynamics of the Notch1 negative regulatory region in autoinhibited states before and after S1 cleavage, in a proteolytically sensitive "on" state, and in a complex with an inhibitory antibody. Conversion to the "on" state leads to accelerated deuteration in the S2 region and in nearby secondary structural elements within the HD. In contrast, complexation with the inhibitory antibody retards deuteration around the S2 site. Together, these studies reveal how S2 site exposure is promoted by receptor activation and suppressed by inhibitory antibodies.

Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors

BackgroundNotch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Principal FindingsHere we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC50 values as low as 5±3 nM and 0.13±0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR “class I” point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare “class II” or “class III” mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.Conclusions/SignificanceAntibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.

Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

BackgroundNotch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.Principal FindingsThe X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity.Conclusions/SignificanceS1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage.

Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL

AbstractProteolytic resistance of Notch prior to ligand binding depends on the structural integrity of a negative regulatory region (NRR) of the receptor that immediately precedes the transmembrane segment. The NRR includes the 3 Lin12/Notch repeats and the juxtamembrane heterodimerization domain, the region of Notch1 most frequently mutated in T-cell acute lymphoblastic leukemia lymphoma (T-ALL). Here, we report the x-ray structure of the Notch1 NRR in its autoinhibited conformation. A key feature of the Notch1 structure that maintains its closed conformation is a conserved hydrophobic plug that sterically occludes the metalloprotease cleavage site. Crystal packing interactions involving a highly conserved, exposed face on the third Lin12/Notch repeat suggest that this site may normally be engaged in intermolecular or intramolecular protein-protein interactions. The majority of known T-ALL–associated point mutations map to residues in the hydrophobic interior of the Notch1 NRR. A novel mutation (H1545P), which alters a residue at the crystal-packing interface, leads to ligand-independent increases in signaling in reporter gene assays despite only mild destabilization of the NRR, suggesting that it releases the autoinhibitory clamp on the heterodimerization domain imposed by the Lin12/Notch repeats. The Notch1 NRR structure should facilitate a search for antibodies or compounds that stabilize the autoinhibited conformation.

Structure of the Notch1 Negative Regulatory Region: Implications for Normal Activation and Pathogenic Signaling in T-ALL

Proteolytic resistance of Notch prior to ligand binding depends on the structural integrity of a negative regulatory region (NRR) of the receptor that immediately precedes the transmembrane segment. The NRR includes the three Lin12/Notch repeats and the juxtamembrane heterodimerization domain, the region of Notch1 most frequently mutated in T cell acute lymphocytic leukemia. Here, we report the X-ray structure of the Notch1 NRR determined to 2.0 Å resolution in its autoinhibited conformation. A key feature of the Notch1 structure that maintains its closed conformation is a conserved hydrophobic plug that sterically occludes the metalloprotease cleavage site. Crystal packing interactions involving a highly conserved, exposed face on the third Lin12/Notch repeat suggest that this site may normally be engaged in inter- or intra-molecular protein-protein interactions. The vast majority of known T-ALL-associated point mutations of human Notch1 map to residues in the hydrophobic interior of the NRR. A novel mutation (H1545P), which alters a residue at the crystal-packing interface, leads to ligand-independent increases in signaling in reporter gene assays despite only mild destabilization of the NRR, suggesting that it releases the autoinhibitory clamp on the heterodimerization domain imposed by the Lin12/Notch repeats. The Notch1 NRR structure should facilitate a search for antibodies or compounds that stabilize the autoinhibited conformation.