RAM-induced allostery facilitates assembly of a notch pathway active transcription complex.
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.
Friedmann
David R
DR
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524, USA.
Wilson
Jeffrey J
JJ
Kovall
Rhett A
RA
D000494:Allosteric Regulation; D000818:Animals; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051880:Receptors, Notch / Q000378:metabolism*; D015398:Signal Transduction; D013816:Thermodynamics; D014158:Transcription, Genetic / Q000235:genetics*
D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D011518:Proto-Oncogene Proteins; D051880:Receptors, Notch; D004247:DNA