title: Structural requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 transcriptional activation complex.
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-Jkappa, 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 beta-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-Jkappa (recombination-signal-sequence-binding protein for Jkappa 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.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Yunsun
authors.initials: Y
authors.lastname: Nam
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Weng
authors.forename: Jon C
authors.initials: JC
authors.lastname: Aster
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Blacklow
mesh_terms: D017487:Ankyrins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055592:Biophysical Phenomena; D001703:Biophysics; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020569:Proto-Oncogene Proteins c-rel / Q000235:genetics; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D015534:Trans-Activators; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000502:physiology*
chemical_list: D017487:Ankyrins; D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C430872:MAML1 protein, human; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; D009687:Nuclear Proteins; D020569:Proto-Oncogene Proteins c-rel; C497570:RBPJ protein, human; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA