Phosphoserine inhibits neighboring arginine methylation in the RKS motif of histone H3

Abstract

The effects of phosphorylation of histone H3 at serine 10 have been studied in the context of other posttranslational modifications such as lysine methylation. We set out to investigate the impact of phosphoserine-10 on arginine-8 methylation. We performed methylation reactions using peptides based on histone H3 that contain a phosphorylated serine and compared the extent of arginine methylation with unmodified peptides. Results obtained via fluorography indicate that peptides containing a phosphorylated serine-10 inhibit deposition of methyl groups to arginine-8 residues. To further explore the effects of phosphoserine on neighboring arginine residues, we physically characterized the non-covalent interactions between histone H3 phosphoserine-10 and arginine-8 using ³¹P NMR spectroscopy. A salt bridge was detected between the negatively charged phosphoserine-10 and the positively charged unmodified arginine-8 residue. This salt bridge was not detected when arginine-8 was symmetrically dimethylated. Finally, molecular simulations not only confirm the presence of a salt bridge but also identify a subset of electrostatic interactions present when arginine is replaced with alanine. Taken together, our work suggests that the negatively charged phosphoserine maximizes its interactions. By limiting its exposure and creating new contacts with neighboring residues, it will inhibit deposition of neighboring methyl groups, not through steric hindrance, but by forming intrapeptide interactions that may mask substrate recognition. Our work provides a mechanistic framework for understanding the role of phosphoserine on nearby amino acid residues and arginine methylation.

Keywords: Histone crosstalk; Methylarginine; Methylation; PRMT; Phosphorylation; Phosphoserine; Post translational modification (PTM); Symmetric arginine dimethylation (SDMA).

Figure 1.

Methylation of peptide 1 (TKQTARKSTGGKAP) or peptide 2 (TKQTARKSpTGGKAP) corresponding to histone H3 detected via fluorography. A) GST-PRMT5 (1μg), B) GST-PRMT4 (1μg), C and D) GST-PRMT1 (1μg), were incubated with substrates histone H3, peptide 1 (1μg) or peptide 2 (1 μg) in the presence of 0.5 μM S-adenosyl-L-[methyl-³H]methionine for 1 hr at 37 °C in a final volume of 30 μl of HEPES buffer as described in Materials and Methods. The samples were then resolved via 20% SDS-PAGE. Full-length histone H3 was used as a positive control, PRMT, and peptide alone reactions lacking enzyme or substrates were used as negative controls. Negative controls such as PRMT1 alone or Peptide 1 alone were carried out in the presence of all reagents, including [³H]AdoMet. The radioactive methylation reactions were exposed on film as described Materials and Methods for A) 8 days, B) 7 days, C) 3 weeks and D) 9 days.

Figure 2.

Hydrolysates of methylation reactions of PRMT1 incubated with either unmodified peptide 1 or phosphorylated peptide 2 and resolved by TLC. TLC for hydrolysates of the reaction mixture and individual and mixed standards of ADMA, MMA, and SDMA. The upper portion shows the ninhydrin staining of the TLC plate containing the arginine derivative standards; the lower portion shows the radioactivity corresponding to the TLC slices of the reaction mixture lane. A) TLC of hydrolysates for PRMT1, 5001 cpms; mix lane contains reaction mixture and standards; B) TLC of hydrolysates for PRMT1 incubated with phosphorylated peptide 2, 1596 cpms; C) TLC of hydrolysates for PRMT1 alone (control), 1702 cpms, and D) TLC of hydrolysates for peptide 1 and peptide 2 alone controls. All controls contained [³H]AdoMet.

Figure 3.

Dependence of the ³¹P NMR chemical shifts of 1 mg of A) Peptide 1 (TKQTARKS(PO₃H₂)TGGKAP), one of three trials, B) Peptide 2 (TKQTARAS(PO₃H₂)TGGKAP), one of three trials, C) Peptide 3 (TKQTAAKS(PO₃H₂)TGGKAP), one of three trials, D) Peptide 4 (TKQTAAAS(PO₃H₂)TGGKAP), one of three trials, E) Peptide 5 (TKQTAR(SDMA)KS(PO₃H₂)TGGKAP), one of two trials, and F) Peptide 6 (TKQTAR(SDMA)AS(PO₃H₂)TGGKAP), one of two trials. All peptides were dissolved in approximately 0.5 ml of 30 mM HEPES, 5 mM MgCl₂, 5 mM KCl, 10 % glycerol and 8 % D₂O at 22 °C. The titration curves were calculated with the parameters described in Materials and Methods. The x-axis is the experimental pH measured and the y-axis is the chemical shift observed at the respective pH value.

Figure 4.

Comparision of histone H3 peptides 1-RKS(PO₃H₂), and 4-AAS(PO₃H₂), (PDB file 1kx5, Python, chain; ribbon). A) Phosphoserine-10 (red/orange), creates a salt bridge with arginine-8 (blue/white). The salt-bridge is denoted by dashed lines and by the the proximity of the atoms (1.92 Å and 2.07 Å , respectively). B) In the absence of positively charged arginine-8 and lysine-9, phosphoserine-10 (red/orange), maintains or creates interactions with other atoms. These include the threonine-11 residue, other lysine residues, and backbone amide protons found both nearby and more distant to phosphoserine-10.