Keywords

Phosphorylation, pThr, Ser/Thr, Protein Conformation, Structural Biology, ACS

Reference

DOI: 10.1021/acschembio.3c00068

Abstract

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. This paper examines structural differences between serine and threonine phosphorylation using circular dichroism (CD), NMR spectroscopy, PDB analysis, X-ray crystallography, and computational methods. Both pSer and pThr show substantial conformational restriction in their dianionic forms. However, phosphothreonine (pThr) displays a stronger disorder-to-order transition, adopting a cyclic conformation stabilized by three key noncovalent interactions:

  1. Intraresidue phosphate-amide hydrogen bond
  2. n → π* interaction between consecutive carbonyls
  3. n → σ* interaction between phosphate oxygen lone pair and C–Hβ antibond, restricting side chain.

Phosphothreonine mimics proline’s cyclization, leading to a compact, ordered conformation, favoring polyproline II helix torsions. These findings highlight how threonine phosphorylation induces larger, switch-like conformational changes, while serine phosphorylation leads to subtler, rheostat-like adjustments.

Notes

1. General Background

  • Ser and Thr account for over 90% of phosphorylation sites in proteomics studies.
  • Ser is more frequently phosphorylated than Thr, but functional distinctions exist:
    • Evolutionary rate differences
    • Interaction preferences
    • Structural impact on proteins.
  • pThr has stronger conformational effects, acting as binary structural switch, while pSer functions as a rheostat-like modulator.

2. Key Findings

2.1 Structural Impact

  • Phosphorylation of Thr induces compact, ordered structures, especially in the dianionic state (pThr²⁻).
  • Three key noncovalent interactions stabilize pThr:
    1. Phosphate-amide hydrogen bond
    2. n → π* between carbonyls
    3. n → σ* between phosphate oxygen and side-chain C–Hβ
  • pThr mimics proline-like cyclic constraints but via noncovalent interactions, unlike Pro’s covalent cyclization.
  • Preferred torsions of pThr:
    • ϕ ∼ −60° (favoring polyproline II and α-helix)
    • χ1 = g⁻; χ2 ∼ +115° (C–H/O–P eclipsed bonds)
  • pThr adopts more ordered conformation than pSer, explaining functional differences in signaling and structural regulation.

2.2 Functional Implications

  • pThr enables sharp, stepwise switching in protein conformation and function — “all-or-nothing” effect.
  • pSer induces subtle, adjustable changes — gradual, tunable regulation.
  • pThr’s rigid cyclic structure facilitates its role in activation loops of kinases and protein-protein interaction sites.

3. Experimental Observations

  • CD spectroscopy: Both pSer and pThr increase PPII content (positive CD band ~220 nm), but pThr has stronger effect.
  • NMR & Crystallography: Confirm cyclic-like conformation of pThr, stabilized by described interactions.
  • Computational modeling: Supports structural restriction upon phosphorylation, especially for pThr²⁻ at physiological pH.
  • Bioinformatics analysis of PDB: Validates presence of pThr’s ordered conformations across many proteins.

4. Conceptual Insights

  • Difference in flexibility:
    • pThr more constrained due to γ-methyl group, favoring a locked, compact structure.
    • pSer lacks γ-methyl group, remains more flexible even when phosphorylated.
  • Functional differences:
    • pThr functions as binary switch (on/off structural transitions).
    • pSer provides graded responses, adjusting conformation dynamically.

5. Speculative / Thoughtful Observations

  • Two concepts to assess size/compactness:
    1. Molecular volume: Ser < Pro < Thr < pSer < pThr
    2. Compactness considering noncovalent locking: Ser < Thr < pSer < Pro < pThr (revised by structural findings here).
  • Proline-like behavior of pThr provides insight into why phosphorylation of Thr can be functionally distinct from Ser, beyond just the presence of the phosphate group.

Take-home Messages

  • Phosphothreonine (pThr) adopts a highly ordered, compact conformation, stabilized by three noncovalent interactions.
  • Structural difference between pThr and pSer underlies their distinct biological roles.
  • pThr can act as a stepwise structural switch, inducing major conformational shifts in proteins.
  • pSer tends to modulate structures more gradually, supporting fine-tuned regulation.
  • Understanding these intrinsic differences helps explain why certain phosphorylation sites are Ser or Thr, and guides design in signaling studies and synthetic biology.