Molecular Basis for Jagged-1/Serrate Ligand Recognition by the Notch Receptor

Pat Whiteman,Beatriz Hernandez De Madrid,Paul Taylor,Demin Li,Rebecca Heslop,Nattnee Viticheep,Joyce Zi Tan,Hideyuki Shimizu,Juliana Callaghan,Massimo Masiero,Ji Liang Li,Alison H Banham,Adrian L Harris,Susan M Lea,Christina Redfield,Martin Baron,Penny A Handford

doi:10.1074/jbc.m112.428854

Pat Whiteman, Beatriz Hernandez De Madrid + Show 15 more

Open Access

https://doi.org/10.1074/jbc.m112.428854

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Abstract

We have mapped a Jagged/Serrate-binding site to specific residues within the 12th EGF domain of human and Drosophila Notch. Two critical residues, involved in a hydrophobic interaction, provide a ligand-binding platform and are adjacent to a Fringe-sensitive residue that modulates Notch activity. Our data suggest that small variations within the binding site fine-tune ligand specificity, which may explain the observed sequence heterogeneity in mammalian Notch paralogues, and should allow the development of paralogue-specific ligand-blocking antibodies. As a proof of principle, we have generated a Notch-1-specific monoclonal antibody that blocks binding, thus paving the way for antibody tools for research and therapeutic applications.

Highlights

The site of Jagged/Serrate ligand recognition by Notch is unknown
We have previously shown that the 12th EGF (EGF12) domain of Notch is directly implicated in ligand binding [14], To investigate this region further, we have made a series of single amino acid substitutions in human and Drosophila Notch designed to probe ligand recognition, while retaining Notch calcium binding, which is already known to be critical for this interaction [15].Using flow cytometry and cell aggregation assays, we have identified a globally conserved hydrophobic binding site that is adjacent to the residue that forms part of the O-fucosylation consensus sequence (C2-X4-(S/T)-C3) in EGF12, where C2 and C3 represent the second and third conserved cysteines [16]
One was similar to wild type (WT) in its ability to bind J-1 (M479A), but the other, L468A, was completely unable to bind J-1 (Fig. 2)

Summary

Background

The site of Jagged/Serrate ligand recognition by Notch is unknown. Results: Two critical residues involved in an intramolecular hydrophobic interaction across the central ␤-sheet of EGF12 form a ligand-binding platform. We have previously shown that the 12th EGF (EGF12) domain of Notch is directly implicated in ligand binding [14], To investigate this region further, we have made a series of single amino acid substitutions in human and Drosophila Notch designed to probe ligand recognition, while retaining Notch calcium binding, which is already known to be critical for this interaction [15].Using flow cytometry and cell aggregation assays, we have identified a globally conserved hydrophobic binding site that is adjacent to the residue that forms part of the O-fucosylation consensus sequence (C2-X4-(S/T)-C3) in EGF12, where C2 and C3 represent the second and third conserved cysteines [16] This residue is reported to be subjected to further modification by the Fringe enzymes [17], resulting in changes to Notch activity [18]. Jagged-1/Serrate Recognition by Notch that it can be exploited to raise paralogue-specific antibodies that block ligand binding

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Equivalent substitution in Drosophila Notch