An Inherent Structural Difference Between Serine and Threonine Phosphorylation: Phosphothreonine Prefers an Ordered, Compact, Cyclic Conformation

Abstract

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms.Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted φ (φ ~ -60˚) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n→π* interaction between consecutive carbonyls, and an n→σ* interaction between the phosphate Oγ lone pair and the antibonding orbital of C-Hβ that restricts the χ 2 side chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic proline's backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are φ,ψ = polyproline helix or α-helix (φ ~ -60˚); χ 1 = g -; χ 2 = eclipsed C-H/O-P bonds. This structural signature is observed in diverse proteins, including the activation loops of protein kinases and protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.

α-Helix stabilization by co-operative side chain charge-reinforced interactions to phosphoserine in a basic kinase-substrate motif

“…There is an inverse correlation between peptide helicity and 3 JPH, with the ahelical peptide 7 having the highest 3 JPH (9 Hz) and peptide 1 having the lowest (5.3 Hz). These values match very well with those found by Pandey et al 36 for pThrand pSer-containing peptides; the high value for peptide 7 suggests a well-defined P-O-C-H dihedral and ordered pThr side chain, whereas the lower values for peptides 3, 5 and 1 are consistent with a greater degree of pSer side chain freedom. The reduced value for 1 compared to 3, 5 and 6 is interesting and suggests altered dihedral propensities for the most helical pSer-containing peptide.…”

“…49 Moreover, recent studies have shown Thr phosphorylation promotes intra-residue hydrogen-bonding between the phosphate and the backbone amide, which would disrupt helix propagation in a manner analogous to the effect of Pro. 36 Taken together these results highlight the secondary structural sequence context of phosphorylation and highlight the potential for it to affect PPIs not only through orthosteric 13 or allosteric 50 changes in recognition features and switching off recognition by changing conformation, 14 but also through phospho-driven stabilization of an α-helical recognition motif.…”

“…This can be attributed to the proclivity of pThr to adopt a compact conformation that is incompatible with a helical conformation and is evidently not countered by the potential benefit of non-covalent interaction with side chains as for pSer (peptide 1). 36…”

α-Helix stabilization by co-operative side chain charge-reinforced interactions to phosphoserine in a basic kinase-substrate motif

“…Moreover, recent studies have shown Thr phosphorylation promotes intra-residue hydrogen-bonding between the phosphate and the backbone amide, which would disrupt helix propagation in a manner analogous to the effect of Pro. 27 Taken together these results highlight the secondary structural sequence context of phosphorylation and highlight the potential for it to affect PPIs not only through orthosteric 13 or allosteric 43 changes in recognition features and switching off recognition by changing conformation, 14 but also through phospho-driven stabilization of an α-helical recognition motif. piperidine: formic acid (75:20:5) solution (6 mL), room temperature method (5 min), wash with DMF (15 mL), add DMF: piperidine: formic acid (75:20:5) solution (6 mL), room temperature method (5 min), wash with DMF (15 mL), clean resin dip tube, wash with DMF (15 mL).…”

“…Such a conformation is incompatible with a helical conformation and is evidently not countered by the benefit of side-chain hydrogen-bonding as for pSer (peptide 1). 27 The effect of pH on helicity for peptide 1 provided useful insights on the contribution of charge-based interactions between side chains (Fig. 3).…”