Phosphorylation versus O-GlcNAcylation: Computational Insights into the Differential Influences of the Two Competitive Post-Translational Modifications.

Abstract

Phosphorylation and O-GlcNAcylation are rapidly cycling intracellular protein post-translational modifications (PTMs) that can compete for the same serine (S) and threonine (T) sites. Limited crystal structure information is available on the direct influence of these PTMs on the underlying protein structure, especially for O-GlcNAcylation. NMR and CD studies show that these competitive-PTMs can have the same or differential influence on the overall secondary structure. In Tau derived peptide fragments, it was found that phosphorylation stabilized PPII conformations while O-GlcNAcylation destabilized the same. In the absence of substantial structural information, we have performed a systematic computational study utilizing PDB analysis, QM calculations, and MD simulations to identify key structural trends upon PTM. Our analysis of the limited PDB data set revealed conformational shifts from PPII to α-helical geometry upon serine phosphorylation and in the opposite direction, from α-helical to PPII geometry upon threonine phosphorylation. Gas phase QM calculations covering the complete Ramachandran ϕ/ψ space using model native, phosphorylated, and O-GlcNAcylated dipeptide systems revealed preferences toward α-helical conformations. However, the major structural transitions were observed in the MD simulations upon the inclusion of solvation. The model dipeptide simulations revealed a preference for PPII and α-helical conformations for phosphorylated serine and threonine, while O-GlcNAcylated dipeptides exhibited a complete shift toward extended conformations, β-sheet and PPII, disfavoring the α-helical conformation. For the Baldwin α-helix simulations, it was found that both phosphorylation and O-GlcNAcylation destabilized the helix; however, the destabilization was governed by H-bonding and electrostatic interactions in the former, while the latter was controlled by hydrophobic collapse and steric interactions. The presence of lysine in close proximity of phosphate leads to potentially stable salt bridge interactions, which can influence the structure on the basis of the relative placement of the lysine with respect to the PTM site. Similar strong lysine-phosphate contacts were observed in the model Tau peptides, which steers the conformations toward PPII geometries, highlighting the direct influence of the PTM on function.