Effects of Charge Interactions and Transient Secondary Structure Elements on the Function of the Disordered RAM Region of the Notch Receptor

Abstract

The Notch signaling pathway is crucial for cellular development. The intracellular domain of the Notch receptor (NICD) contains the 140-residue intrinsically disordered RAM region followed by seven ankyrin repeats, a nuclear localization sequence, and a C-terminal degradation sequence. The RAM N-terminus and the ankyrin repeats of NICD form a bivalent interaction with the transcription factor CSL that leads to the activation of Notch target genes. Assays measuring transcription activity of NICD variants containing sequence deletions and insertions in the region of RAM between the N-terminal binding site and the ankyrin repeats reveal that RAM mostly behaves as a disordered statistical coil. However, deviations from this model suggest that NICD bivalency is affected by sequence-specific elements within RAM, such as transient secondary structure elements and/or charge interactions. NMR spectroscopy has revealed three regions of RAM with helical propensity and two regions that are extended and dynamic. Transcription assays show that sequence substitutions to these regions have a modest effect on transcriptional activation. In contrast, charge interactions within the RAM are crucial for transcriptional activation. Redistributing the charged residues within RAM changes the tertiary structure of RAM and decreases transcriptional activation. NICD variants with highly segregated positive and negative charges in the RAM region are more compact than wild-type RAM and display almost no transcriptional activation. Variants containing oppositely charged residues that are more mixed than in wild-type RAM are more extended and exhibit smaller decreases in transcriptional activation. Current work is focused on understanding why compaction of the RAM region affects Notch transcriptional activation. Understanding how the secondary and tertiary structure elements of RAM affect transcriptional activation will provide insight into the general roles of IDPs in signaling pathways.