This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our project has two goals: one is to determine how Notch receptors are maintained in a resting conformation prior to the induction of signaling by ligands, and the other is to determine the structure of transcription complexes formed by intracellular Notch. Notch plays a key role in cell fate decisions throughout development. Notch signals are normally induced when a ligand of the DSL family expressed on one cell binds to a Notch receptor on a neighboring cell. The negative regulatory region of Notch receptors, which consists of a series of Notch-unique LIN12/Notch (LNR) repeats and a novel domain called HD (for heterodimerization region), maintains Notch receptors in their resting conformation, and we are working on solving the structures of the negative regulatory regions of different Notch receptors. The only known effector functions of activated Notch are mediated by the transcriptional complex. We have crystals that include CSL (DNA-binding protein), Notch (ankyrin repeats and RAM region), Mastermind (co-activator) on DNA. We have previously solved the structure of this complex without the RAM region (Nam et al., Cell 2006), and we now have incorporated the additional key peptide portion of RAM to determine where this Notch-specific domain binds in the complex. We are working on improving the crystals of complexes that include the RAM domain to build a good quality atomic model of complexes that include the RAM region. Moreover, we intend to incorporate other domains that compete with RAM to bind CSL into the complex as well. Determining the structures of these complexes will shed light on the mechanism by which Notch binding switches CSL from a repressor to an activator.
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