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, Stephen C. Blacklow

Research output: Contribution to journalArticlepeer-review

110 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)4381-4390
Number of pages10
JournalBlood
Volume113
Issue number18
DOIs
StatePublished - 2009

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