Control of transcriptional activity by design of charge patterning in the intrinsically disordered RAM region of the Notch receptor
Edited by Ken A. Dill, Stony Brook University, Stony Brook, NY, and approved September 20, 2017 (received for review April 12, 2017)
Significance
Charge patterning is a key feature of intrinsically disordered protein regions. Here we test whether charge pattering is important for biochemical and biological function, using the “RAM” disordered region of the Notch receptor. The Notch signaling pathway is important in stem-cell biology and cancer. Using computer design, we built 13 charge permutants that span a broad range of charge segregation. These permutants have profound effects on conformational properties, binding affinity to the downstream transcription factor, CSL, and potency in transcriptional activation. WT Notch has the optimal segregation value for activation, whereas higher levels of segregation disrupt binding and activation. Our study paves the way for control of biological function through redesign of charge patterning.
Abstract
Intrinsically disordered regions (IDRs) play important roles in proteins that regulate gene expression. A prominent example is the intracellular domain of the Notch receptor (NICD), which regulates the transcription of Notch-responsive genes. The NICD sequence includes an intrinsically disordered RAM region and a conserved ankyrin (ANK) domain. The 111-residue RAM region mediates bivalent interactions of NICD with the transcription factor CSL. Although the sequence of RAM is poorly conserved, the linear patterning of oppositely charged residues shows minimal variation. The conformational properties of polyampholytic IDRs are governed as much by linear charge patterning as by overall charge content. Here, we used sequence design to assess how changing the charge patterning within RAM affects its conformational properties, the affinity of NICD to CSL, and Notch transcriptional activity. Increased segregation of oppositely charged residues leads to linear decreases in the global dimensions of RAM and decreases the affinity of a construct including a C-terminal ANK domain (RAMANK) for CSL. Increasing charge segregation from WT RAM sharply decreases transcriptional activation for all permutants. Activation also decreases for some, but not all, permutants with low charge segregation, although there is considerable variation. Our results suggest that the RAM linker is more than a passive tether, contributing local and/or long-range sequence features that modulate interactions within NICD and with downstream components of the Notch pathway. We propose that sequence features within IDRs have evolved to ensure an optimal balance of sequence-encoded conformational properties, interaction strengths, and cellular activities.
Acknowledgments
We thank current and past members of the D.B. and R.V.P. laboratories, specifically Christine Hatem, Scott Johnson, Alex Holehouse, Tyler Harmon, Kiersten Ruff, and Andreas Vitalis, for helpful discussions and technical assistance and the Johns Hopkins University Center for Molecular Biophysics for providing facilities and resources. This work was supported by Grant GM060001 from the National Institutes of Health (to D.B.) and Grants MCB 1121867 and MCB 1614766 from the National Science Foundation (to R.V.P.).
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Cover image: Pictured is a developing Arabidopsis thaliana lateral root labeled with a fluorescent transcriptional reporter, with cell outlines shown in blue. Yujuan Du and Ben Scheres found that the PLETHORA transcription factors PLT3, PLT5, and PLT7 regulate the initiation of cell divisions at the start of Arabidopsis lateral root development. According to the authors, PLETHORA transcription factors are key molecular triggers for the establishment of new meristems and tissue differentiation associated with a new growth axis. See the article by Yujuan Du and Ben Scheres on pages 11709–11714. Image courtesy of Yujuan Du.
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© 2017. Published under the PNAS license.
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Published online: October 12, 2017
Published in issue: October 31, 2017
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Acknowledgments
We thank current and past members of the D.B. and R.V.P. laboratories, specifically Christine Hatem, Scott Johnson, Alex Holehouse, Tyler Harmon, Kiersten Ruff, and Andreas Vitalis, for helpful discussions and technical assistance and the Johns Hopkins University Center for Molecular Biophysics for providing facilities and resources. This work was supported by Grant GM060001 from the National Institutes of Health (to D.B.) and Grants MCB 1121867 and MCB 1614766 from the National Science Foundation (to R.V.P.).
Notes
This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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