Journal of Biomolecular NMR

, Volume 55, Issue 4, pp 323–337 | Cite as

Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

Article

Abstract

The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1H,15N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

Keywords

NMR spectroscopy Post-translational modifications Phosphorylation Protein Pin1 WW binding module 

Abbreviations

AD

Alzheimer’s disease

IDPs

Intrinsically disordered proteins

Pin1

Protein interacting with NIMA-1

NIMA

Never in mitosis gene A

PKA

Protein kinase A

MALDI-TOF MS

Matrix-assisted laser desorption ionization- time of flight mass spectrometry

PTMs

Post translational modifications

RP-HPLC

Reverse phase high pressure liquid chromatography

WW

Trp–Trp binding module

WWPKA

PKA-phosphorylated WW domain

Phospho-WW

Phosphorylated form of the WW domain

Supplementary material

10858_2013_9716_MOESM1_ESM.docx (375 kb)
Supplementary material 1 (DOCX 374 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Caroline Smet-Nocca
    • 1
    • 2
  • Hélène Launay
    • 1
  • Jean-Michel Wieruszeski
    • 1
  • Guy Lippens
    • 1
  • Isabelle Landrieu
    • 1
    • 3
  1. 1.Institut Fédératif de Recherches 147, CNRS UMR 8576Université de Lille-Nord de FranceVilleneuve d’AscqFrance
  2. 2.Unité de Glycobiologie Structurale et Fonctionnelle (CNRS UMR 8576)Université de Lille-Nord de FranceVilleneuve d’AscqFrance
  3. 3.Unité de Glycobiologie Structurale et Fonctionnelle (CNRS UMR 8576), Interdisciplinary Research InstituteUniversité de Lille-Nord de FranceVilleneuve d’AscqFrance

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