Structural requirements for assembly of the CSL·intracellular Notch1·Mastermind-like 1 transcriptional activation complex

Yunsun Nam, Andrew P. Weng, Jon C. Aster, Stephen C. Blacklow

Research output: Contribution to journalArticle

152 Scopus citations

Abstract

Ligand binding by Notch receptors triggers a series of proteolytic cleavages that liberate the intracellular portion of Notch (ICN) from the cell membrane, permitting it to translocate to the nucleus. Nuclear ICN binds to a highly conserved DNA-binding transcription factor called CSL (also known as RBP-Jκ, CBF1, Suppressor of Hairless, and Lag-1) and recruits Mastermind-like transcriptional co-activators to form a transcriptional activation complex. Using bioinformatics tools, we identified a Rel homology region (RHR) within CSL that was used as a guide to determine the minimal protein requirements for ternary complex formation. The RHR of CSL contains both the N- and C-terminal β-sheet domains (RHR-n and RHR-c) of typical Rel transcription factors, as judged by circular dichroism spectra. Binding of monomeric CSL to DNA requires the entire RHR of CSL and an additional 125-residue N-terminal sequence, whereas binding to ICN requires only the RHR-n domain. Although the RAM (RBP-Jκ (recombination-signal-sequence-binding protein for Jκ genes)-associated molecule) domain of ICN is flexible and relatively unstructured as an isolated polypeptide in solution, it associates stably with CSL on DNA. Recruitment of Mastermind-like 1 (MAML1) to CSL·ICN complexes on DNA requires inclusion of the ankyrin repeat domain of ICN, and N- and C-terminal sequences of CSL extending beyond the DNA-binding region. The requirement for cooperative assembly of the MAML1·ICN·CSL·DNA complex suggests that a primary function of ICN is to render CSL competent for MAML loading. On the basis of our results, we present a working structural model for the organization of the MAML1·ICN·CSL·DNA complex.

Original languageEnglish (US)
Pages (from-to)21232-21239
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number23
DOIs
StatePublished - Jun 6 2003

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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