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{phi} ({phi} [~] -60{degrees}) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n[->]{pi}* interaction between consecutive carbonyls, and an n[->]{sigma}* interaction between the phosphate O{gamma} lone pair and the antibonding orbital of C-H{beta} that restricts the{chi} 2 side chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic prolines backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are{phi} ,{psi} = polyproline helix or -helix ({phi} [~] -60{degrees});{chi} 1 = g-;{chi} 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.