title: Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL.
abstract: 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 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.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Wendy R
authors.initials: WR
authors.lastname: Gordon
authors.forename: Monideepa
authors.initials: M
authors.lastname: Roy
authors.forename: Didem
authors.initials: D
authors.lastname: Vardar-Ulu
authors.forename: Megan
authors.initials: M
authors.lastname: Garfinkel
authors.forename: Marc R
authors.initials: MR
authors.lastname: Mansour
authors.forename: Jon C
authors.initials: JC
authors.lastname: Aster
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Blacklow
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D018360:Crystallography, X-Ray; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051883:Receptor, Notch2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D008156:Luciferases