Quantitative dissection of the Notch:CSL interaction: insights into the Notch-mediated transcriptional switch.
Complex formation between the intracellular domain of the Notch receptor (NICD) and the transcription factor CSL is indispensable for transcriptional activation. To understand how NICD displaces CSL-associated co-repressors, we have quantified the binding of different Notch1 ICD regions to a key interaction domain (the beta trefoil domain, or BTD) of human CSL. Electrophoresis, scattering, and titration calorimetry indicate that NICD and BTD combine to form a 1:1 heterodimer. Neither the Notch1 ankyrin domain (ANK) nor C-terminal region contributes binding energy towards BTD. In contrast, binding energy is attributed largely to a short segment including the conserved WFP sequence motif within the RAM region (the approximately 140 residue polypeptide segment N-terminal to the ANK domain); substitution of this motif substantially reduces affinity. Short (< or =25 residues) WFP-containing peptides encoded by the four mammalian Notch genes have similar affinities to BTD; thus, activity differences between paralogues either result from other regions of NICD and CSL or from differences in interaction with downstream components. The importance of RAM was demonstrated by the ability of a short RAM peptides to dissociate NICD:CSL interaction in cellular lysates. These results support an emerging molecular mechanism for the displacement of co-repressors from DNA-bound CSL by NICD.
Lubman
Olga Y
OY
T.C Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
Ilagan
Ma Xenia G
MX
Kopan
Raphael
R
Barrick
Doug
D
D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D002457:Cell Extracts; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch / Q000737:chemistry / Q000378:metabolism*; D017385:Sequence Homology; D012996:Solutions; D013816:Thermodynamics; D014158:Transcription, Genetic
D002457:Cell Extracts; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D010455:Peptides; C497570:RBPJ protein, human; D051880:Receptors, Notch; D012996:Solutions