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 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.
Gordon
Wendy R
WR
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
Roy
Monideepa
M
Vardar-Ulu
Didem
D
Garfinkel
Megan
M
Mansour
Marc R
MR
Aster
Jon C
JC
Blacklow
Stephen C
SC
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
C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D008156:Luciferases