RAM-induced Allostery Facilitates Assembly of a Notch Pathway Active Transcription Complex

摘要

The Notch pathway is a conserved cell-to-cell signaling mechanism, in which extracellular signals are transduced into transcriptional outputs through the nuclear effector CSL. CSL is converted from a repressor to an activator through the formation of the CSL-NotchIC-Mastermind ternary complex. The RAM (RBP-J associated molecule) domain of NotchIC avidly interacts with CSL; however, its role in assembly of the CSL-NotchIC-Mastermind ternary complex is not understood. Here we provide a comprehensive thermodynamic, structural, and biochemical analysis of the RAM-CSL interaction for components from both mouse and worm. Our binding data show that RAM and CSL form a high affinity complex in the presence or absence of DNA. Our structural studies reveal a striking distal conformational change in CSL upon RAM binding, which creates a docking site for Mastermind to bind to the complex. Finally, we show that the addition of a RAM peptide in trans facilitates formation of the CSL-NotchIC-Mastermind ternary complex in vitro. The Notch pathway is a conserved cell-to-cell signaling mechanism, in which extracellular signals are transduced into transcriptional outputs through the nuclear effector CSL. CSL is converted from a repressor to an activator through the formation of the CSL-NotchIC-Mastermind ternary complex. The RAM (RBP-J associated molecule) domain of NotchIC avidly interacts with CSL; however, its role in assembly of the CSL-NotchIC-Mastermind ternary complex is not understood. Here we provide a comprehensive thermodynamic, structural, and biochemical analysis of the RAM-CSL interaction for components from both mouse and worm. Our binding data show that RAM and CSL form a high affinity complex in the presence or absence of DNA. Our structural studies reveal a striking distal conformational change in CSL upon RAM binding, which creates a docking site for Mastermind to bind to the complex. Finally, we show that the addition of a RAM peptide in trans facilitates formation of the CSL-NotchIC-Mastermind ternary complex in vitro. The Notch pathway mediates signaling between neighboring cells and plays important roles in cellular differentiation, proliferation, apoptosis, and stem cell renewal (1Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4951) Google Scholar, 2Bonde J. Hess D.A. Nolta J.A. Curr. Opin. Hematol. 2004; 11: 392-398Crossref PubMed Scopus (34) Google Scholar). Notch signaling is required during embryonic development and patterning as well as for cell fate decisions during adult homeostasis. Proper regulation of Notch is essential, because errant signaling results in congenital defects, cardiovascular disorders, and cancer (3Gridley T. Hum. Mol. Genet. 2003; 12: R9-R13Crossref PubMed Google Scholar, 4Garg V. Muth A.N. Ransom J.F. Schluterman M.K. Barnes R. King I.N. Grossfeld P.D. Srivastava D. Nature. 2005; 437: 270-274Crossref PubMed Scopus (1140) Google Scholar, 5Leong K.G. Karsan A. Blood. 2006; 107: 2223-2233Crossref PubMed Scopus (443) Google Scholar). As first identified and characterized in Drosophila melanogaster and Caenorhabditis elegans (6Greenwald I. Genes Dev. 1998; 12: 1751-1762Crossref PubMed Scopus (460) Google Scholar), the canonical Notch signaling pathway is activated when the cell surface ligand DSL (Delta, Serrate, Lag-2) interacts with the receptor Notch on an adjacent cell (7Bray S.J. Nat. Rev. Mol. Cell Biol. 2006; 7: 678-689Crossref PubMed Scopus (1994) Google Scholar). This receptor-ligand interaction initiates two proteolytic cleavages of the receptor Notch, whereupon the intracellular domain of Notch (NotchIC) 3The abbreviations used are: NotchIC, intracellular domain of Notch; NTD, N-terminal domain; BTD, β-trefoil domain; CTD, C-terminal domain; EMSA, electrophoretic mobility shift assay(s); ITC, isothermal titration calorimetry; GST, glutathione S-transferase; RMSD, root mean square deviation; ANK, ankyrin repeat; RAM, RBP-J associated molecule. i