Keywords

IDR, Biophysics, Proline, Phosphorylation, Proline Isomerization, Compensatory Conformation, Signaling

Reference

DOI: 10.1021/jacs.6b10272

Abstract

Intrinsically disordered regions (IDRs) regulate signaling via multisite phosphorylation, but how phosphorylation affects their conformational properties remains elusive.
Here, an 81-residue IDR from yeast Ash1 is examined using biophysical techniques and molecular simulations, revealing that multisite phosphorylation does not dramatically alter global dimensions, despite the expectation of expansion due to charge addition.
Instead, compensatory local conformational changes, particularly around phosphorylation sites, maintain the overall coil-like, expanded ensemble. Proline and charge patterning emerge as crucial determinants, driving both pre- and post-phosphorylation conformations.

Notes

1. Key Insight: Expanded Coil-like Ensemble Maintained upon Phosphorylation

  • Unphosphorylated Ash1 IDR:
    • Expanded, coil-like conformations with Rg ≈ 28.5 ± 3.4 Å (SAXS).
  • Phosphorylated Ash1 (pAsh1):
    • Surprisingly similar Rg ≈ 27.5 ± 1.2 Å, no global expansion.
  • !Fancy point: Despite adding 10 phosphorylations (charges), global dimensions remain unchanged — an elegant compensatory balance.

2. Sequence Patterning Drives Compensatory Mechanism

  • Proline and charged residues are evenly distributed, counterbalancing local expansions and compactions.
  • Shuffling these residues disrupts this balance, leading to altered Rg, demonstrating sequence-defined control.
  • 🌿 Beautiful sequence design: The IDR “knows” how to stay balanced, thanks to proline and charge patterning.

3. Phosphorylation Effects: Local but Not Global

  • NMR: Local conformational changes detected around phosphorylated residues, including hydrogen bonding and chemical shift changes.
  • Increase in α-helical content in specific regions (residues 430–435 and 470–480) upon phosphorylation — localized structural adaptation.
  • Salt sensitivity tests (NaCl 75–1500 mM): No significant change in Rg, confirming that long-range electrostatics are not dominant in shaping IDR dimensions.

4. Biophysical Takeaways: A Strong Polyampholyte Remains Balanced

  • Phosphorylation converts Ash1 IDR from a weak polyelectrolyte to a strong polyampholyte, but balanced attractive/repulsive interactions maintain conformational properties.
  • Global stability arises from local compensatory shifts, preserving functional conformation.
  • !Awesome: Nature’s way of maintaining “form within flux” — a stabilized dynamic ensemble for signaling precision.

5. Conceptual Innovations and Generalizable Principles

  • The patterning of proline and charges defines IDR conformational behavior before and after multisite phosphorylation.
  • This design principle likely applies to many other IDRs functioning in signaling and multisite phosphorylation cascades.
  • Metaphor of “writers, readers, erasers”: Kinases (writers) impose phosphorylation, IDRs adjust conformation; readers recognize it, and phosphatases (erasers) reset.
  • !Fancy highlight: IDRs use conformational neutrality to maintain flexibility and readiness — a subtle strategy for responsive signaling.

6. Experimental and Theoretical Approaches Combined

  • Small-angle X-ray scattering (SAXS): Revealed global dimensions and lack of compaction/expansion.
  • NMR: Detailed local conformational changes.
  • Molecular simulations: Supported observations of compensatory interactions and conformational balance.

Take-home Messages

  • IDRs like Ash1 can maintain global dimensions upon multisite phosphorylation due to sequence-encoded compensatory interactions.
  • Proline and charge patterning play critical roles in governing these conformational properties, emphasizing design principles in IDR biology.
  • Local structural adjustments accommodate modifications like phosphorylation without disrupting the overall functional ensemble.
  • Understanding these mechanisms is vital for decoding how IDRs mediate signaling, enabling fast yet controlled responses in cellular contexts.

Final Thought: Intrinsic disorder isn’t chaos — it’s a sophisticated balance, a finely tuned ensemble ready for signaling commands.