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The Notch signaling pathway is a cell-cell communication network giving rise to cell differentiation during metazoan development. Activation of the pathway releases the intracellular portion of the Notch receptor to translocate to the nucleus, where it is able to interact with the effector transcription factor CSL, converting CSL from a transcriptional repressor to an activator. This conversion is dependent upon the high affinity binding of the RAM region of the Notch receptor to the β-trefoil domain (BTD) of CSL. Here we probe the energetics of binding to BTD of each conserved residue of RAM through the use of isothermal titration calorimetry and single residue substitution. We find that although the highly conserved ΦWΦP motif is the largest determinant of binding, energetically significant interactions are contributed by N-terminal residues, including a conserved Arg/Lys-rich region. Additionally, we present a …