OGlcNAcylation and Phosphorylation Have Opposing Structural Effects in tau: Phosphothreonine Induces Particular Conformational Order

Abstract

Phosphorylation and OGlcNAcylation are dynamic intracellular protein post-translational modifications that frequently are alternatively observed on the same serine and threonine residues. Phosphorylation and OGlcNAcylation commonly occur in natively disordered regions of proteins, and often have opposing functional effects. In the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of Alzheimer’s disease, whereas OGlcNAcylation stabilizes the soluble form of tau. A series of peptides derived from the proline-rich domain (residues 174–251) of tau was synthesized, with free Ser/Thr hydroxyls, phosphorylated Ser/Thr (pSer/pThr), OGlcNAcylated Ser/Thr, and diethylphosphorylated Ser/Thr. Phosphorylation and OGlcNAcylation were found by CD and NMR to have opposing structural effects on polyproline helix (PPII) formation, with phosphorylation favoring PPII, OGlcNAcylation opposing PPII, and the free hydroxyls intermediate in structure, and with phosphorylation structural effects greater than OGlcNAcylation. For tau196–209, phosphorylation and OGlcNAcylation had similar structural effects, opposing a nascent α-helix. Phosphomimic Glu exhibited PPII-favoring structural effects. Structural changes due to Thr phosphorylation were greater than those of Ser phosphorylation or Glu, with particular conformational restriction as the dianion, with mean 3JαN = 3.5 Hz (pThr) versus 5.4 Hz (pSer), compared to 7.2, 6.8, and 6.2 Hz for Thr, Ser, and Glu, respectively, values that correlate with the backbone torsion angle ϕ. Dianionic phosphothreonine induced strong phosphothreonine amide protection and downfield amide chemical shifts (δmean = 9.63 ppm), consistent with formation of a stable phosphate-amide hydrogen bond. These data suggest potentially greater structural importance of threonine phosphorylation than serine phosphorylation due to larger induced structural effects.

A Phosphorylation‐Induced Turn Defines the Alzheimer’s Disease AT8 Antibody Epitope on the Tau Protein

“…[17] We recently employed MD simulations to probe the structural role of phosphorylation on the AT180 epitope of the tau(208-324) fragment. [19] By applying as imilar strategy,w e generated a1m st rajectory for peptide 1 with phosphorylations at Ser 202 and Thr 205 .I nc ontrast to what was found for other phosphorylated peptides of tau, [20] we observed no significant polyproline II (PPII) content in the secondary structure analyses of the trajectory ( Figure S4). [19] By applying as imilar strategy,w e generated a1m st rajectory for peptide 1 with phosphorylations at Ser 202 and Thr 205 .I nc ontrast to what was found for other phosphorylated peptides of tau, [20] we observed no significant polyproline II (PPII) content in the secondary structure analyses of the trajectory ( Figure S4).…”

OGlcNAcylation and Phosphorylation Have Similar Structural Effects in α-Helices: Post-Translational Modifications as Inducible Start and Stop Signals in α-Helices, with Greater Structural Effects on Threonine Modification

“…21,29,117,118 Both phosphoserine and phosphothreonine exhibited conformations distinct from expected random coil values ( 3 J αN ∼ 6–8 Hz) observed for nonphosphorylated residues in multiple proline-rich peptide contexts, and in particular, phosphothreonine and phosphoserine had greater conformational restriction than the standard phosphomimic Glu (Glu 3 J αN = 5.8–6.3 Hz). Notably, the side chain–main chain hydrogen bonding previously observed, by us and others, would be highly disruptive within α-helices.…”

“…29 On the basis of a parametrized Karplus relationship for P–H three-bond coupling constants, the expected 3 J HβP for an eclipsing interaction is 10.6 Hz. 98 All phosphothreonine-containing peptides were analyzed by 31 P NMR (Table 3).…”

Tunable Control of Polyproline Helix (PPII) Structure via Aromatic Electronic Effects: An Electronic Switch of Polyproline Helix

“…79,80 The basis of this switch is the modification of a residue with poor PPII propensity (Ser or Thr) to a residue with substantially greater PPII propensity (phosphoserine or phosphothreonine). Herein, we demonstrate the ability to use aromatic electronics to control polyproline helix conformation.…”