title: 14-3-3zeta C-terminal stretch changes its conformation upon ligand binding and phosphorylation at Thr232.
abstract: 14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3. Time-resolved fluorescence and molecular dynamics were used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at Thr232, a casein kinase I phosphorylation site located within this region. A tryptophan residue placed at position 242 was exploited as an intrinsic fluorescence probe of the C-terminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3zeta C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3zeta at Thr232 resulted in inhibition of phosphopeptide binding and suppression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of Trp242 also revealed that phosphorylation at Thr232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamic simulations suggest that phosphorylation at Thr232 induces a more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that the conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties.
authors: Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague, Czech Republic.
authors: Veronika
authors: V
authors: Obsilova
authors: Petr
authors: P
authors: Herman
authors: Jaroslav
authors: J
authors: Vecer
authors: Miroslav
authors: M
authors: Sulc
authors: Jan
authors: J
authors: Teisinger
authors: Tomas
authors: T
authors: Obsil
mesh_terms: D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013912:Threonine; D014446:Tyrosine 3-Monooxygenase