An Inherent Difference between Serine and Threonine Phosphorylation: Phosphothreonine Strongly Prefers a Highly 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 χ2 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 II helix > α-helix (ϕ ∼ −60°); χ1 = g –; χ2 ∼ +115° (eclipsed C–H/O–P bonds). This structural signature is observed in diverse proteins, including in the activation loops of protein kinases and in 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.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation

“…Urea strongly promotes PPII, which has led to proposals that PPII is a major conformation in the denatured states of proteins. ,,− The CD spectrum of this peptide in 8 M urea exhibited a much stronger CD signature of PPII, consistent with prior data that urea promotes PPII, including in the Ac-GPPXPPGY-NH 2 peptide host–guest context (Figure S8 of reference ).…”

Charged Amino Acids in the Transmembrane Helix Strongly Affect the Enzyme Activity of Aromatase

“…In the absence of helix-stabilizing hydrogen bonds, pT146 could also induce a kink in helix C that could be regulated by kinase/phosphatase activities (in contrast to the P146 kink of the wild-type isoform). The most common phosphorylation sites in proteins are serine and threonine residues [35]. Although proline codons (CCN) should mutate to serine (UCN) or threonine (ACN) codons with equal probability, we did not find any S146 mutants.…”

“…Another aspect of T146 phosphorylation is the conformational change it induces in the side chain; due to non-covalent interactions of the phosphate, the p-threonine (but not the p-serine) side chain prefers a cyclic conformation similar to the proline side chain [35]. In the absence of helix-stabilizing hydrogen bonds, pT146 could also induce a kink in helix C that could be regulated by kinase/phosphatase activities (in contrast to the P146 kink of the wild-type isoform).…”

Genetic Encoding of Phosphorylated Amino Acids into Proteins

“…Further, threonine is phosphorylated by kinases more slowly than pSer, and pThr is more rapidly degraded by phosphatases, explaining why pThr is much less commonly observed than pSer and implying that pThr is a more transient modification than pSer . Recently, Pandey et al concluded that the side chain of pThr adopts a more conformationally limited and rigid structure than pSer that mimics the backbone cyclization of prolines . Yet, in the few cases where pSer ( 2 ) (and nhpSer ( 20 )) were encoded at threonine phosphorylation sites as a pThr ( 4 ) substitute, they provided better mimicry than Asp/Glu. ,, In 2017, the first pThr GCE system was reported in E. coli , but overall, less effort has gone into pThr systems development compared to pSer, and the pThr systems have not yet seen as much use in addressing biological questions.…”