Submitted August 18, 2008
Accepted December 9, 2008
Structure of the Notch1 negative regulatory region: Implications for normal activation and pathogenic signaling in T-ALL
Wendy R. Gordon, Monideepa Roy, Didem Vardar-Ulu, Megan Garfinkel, Marc R Mansour, Jon C Aster, and Stephen C Blacklow*
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States
Department of Chemistry, Wellesley College, Wellesley, MA, United States
Department of Haematology, Cancer Institute, University College London, London, United Kingdom
* Corresponding author; email: sblacklow{at}rics.bwh.harvard.edu.
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 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 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.
