Hyperphosphorylation of Human Osteopontin and Its Impact on Structural Dynamics and Molecular Recognition

Abstract

Protein phosphorylation is an abundant post-translational modification (PTM) and an essential modulator of protein functionality in living cells. Intrinsically disordered proteins (IDPs) are particular targets of PTM protein kinases due to their involvement in fundamental protein interaction networks. Despite their dynamic nature, IDPs are far from having random-coil conformations but exhibit significant structural heterogeneity. Changes in the molecular environment, most prominently in the form of PTM via phosphorylation, can modulate these structural features. Therefore, how phosphorylation events can alter conformational ensembles of IDPs and their interactions with binding partners is of great interest. Here we study the effects of hyperphosphorylation on the IDP osteopontin (OPN), an extracellular target of the Fam20C kinase. We report a full characterization of the phosphorylation sites of OPN using a combined nuclear magnetic resonance/mass spectrometry approach and provide evidence for an increase in the local flexibility of highly phosphorylated regions and the ensuing overall structural elongation. Our study emphasizes the simultaneous importance of electrostatic and hydrophobic interactions in the formation of compact substates in IDPs and their relevance for molecular recognition events.

Figure 1

Scheme of OPN residues phosphorylated in vitro by Fam20C, identified by MS and NMR spectroscopy. White circles represent previously identified phosphorylation sites.^, Blue and red circles indicate the phosphorylation sites newly identified by MS and NMR spectroscopy, respectively. The blue and red bars indicate the coverage of MS and H^N NMR assignments, respectively.

Figure 2

NMR fingerprint of OPN hyperphosphorylation. (A) ¹H–¹⁵N HSQC NMR spectra of OPN before (black) and after (red) phosphorylation by Fam20C. Note how many serine residues [δ (¹⁵N) ≈116 ppm] experience a downfield shift in the ¹H dimension. (B) Close-up of the serine region of ¹H–¹⁵N HSQC NMR spectra of OPN before (black) and after (red) phosphorylation by Fam20C. The protein sequence with the S-x-E/pS sites colored red is shown in the top left corner. The signal peptide, which is not present in our construct, is colored gray.

Figure 3

¹⁵N NMR relaxation data of OPN (A) before and (B) after phosphorylation, measured at 18.8 T. A charge plot of the protein sequence is shown at the top. Yellow circles indicate the identified phosphorylated residues. ¹⁵N R₁, ¹⁵N R₂, and ¹⁵N–{¹H} NOE relaxation parameters, from top to bottom, respectively, of OPN measured at 293 K. Error bars indicate the fitting errors (¹⁵N R₁ and ¹⁵N R₂) and the error propagation of intensity ratios based on the noise level (hetNOE).

Figure 4

Effect of phosphorylation on long-range interactions measured by PRE experiments. (A) ¹H^N Γ₂ PRE profiles of different OPN cysteine mutants obtained from ¹H^NT₂ NMR experiments. (B) ¹H^N Γ² PRE rates of the phosphorylated OPN mutants D130C (top) and T185C (bottom) determined from ¹H^NT₂ NMR experiments. (C) Plot of the PRE rate difference of OPN and phosphorylated OPN mutants D130C (top) and T185C (bottom). Orange bars indicate the respective mutated cysteine residue with the attached spin-label.

Figure 5

Binding of (phosphorylated) OPN to heparin, monitored by NMR titrations. ¹H–¹⁵N HSQC NMR spectra in the presence of increasing amounts of heparin (red to blue) for the (A) unphosphorylated and (B) hyperphosphorylated forms. Chemical shift perturbations (bottom panel) and fitted K_D of binding regions (middle panel in blue) and the uncompacted region (middle panel in pink) plotted against the residue numbers of (C) OPN and (D) phosphorylated OPN. The corresponding charge plots are shown at the top. Yellow circles indicate the identified phosphorylated residues. The grayscale (from white to black) represents the increasing OPN:hep molar ratio from 1:0.2 to 1:10 (unphosphorylated) and 1:20 (phosphorylated).

Figure 6

Pearson correlation maps of (A) H. sapiens OPN determined from nine PRE profiles and (B) C. japonica OPN determined from 10 PRE profiles. The maps show correlated (red to orange), uncorrelated (light yellow to light blue), and anticorrelated (light blue to dark blue) structural fluctuations. The dashed squares enclose regions of distinct structural compaction. The orange dots represent the spin-label sites. The data for the C. japonica OPN correlation matrix were previously published. Corresponding charge plots are shown at the top.