Phosphorylation-Induced Structural Changes in the C-Terminus of c-Fos Detected by CD and NMR

Abstract

The major modification of the c-Fos protein involves serine phosphoesterification at the sites located at the extreme C-terminus. Several transcriptionally important protein–protein interactions involving c-Fos are mediated by the C-terminus domain phosphorylation. A series of peptides and phosphopeptides corresponding to C-terminus (359–380) of c-Fos protein were synthesized and their conformations were determined by CD spectroscopy and 1H-NMR spectroscopy. The nonphosphorylated peptide (Fos-C) adopts an essentially random-coil conformation in aqueous solution as shown by CD spectroscopy, but becomes slightly more ordered into a type I turn by adding up to 80% trifluoroethanol (TFE) to increase the solvent hydrophobicity. There is tentative NMR evidence for a small population of species containing a type I turn at residues SPTL in the Fos-C. Phosphorylation at S362 induces local conformational changes that can be attributed to the formation of local hydrogen bonds between the phosphate group and nearby amide protons, including additional medium-range (e-HN of R359) effects. Upon phosphorylation at S374, a hydrogen bond between the amide proton and phosphate group of pS374 is formed. However, the most significant changes upon phosphorylation at S374 are the destabilization of this turn structure, which is strongly supported by CD and NMR spectroscopy.