Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

• Published in: PloS one 2015-08, Vol.10 (8), p.e0135976-e0135976
• Main Authors: Ghosh, Anirban, Ratha, Bhisma Narayan, Gayen, Nilanjan, Mroue, Kamal H, Kar, Rajiv K, Mandal, Atin K, Bhunia, Anirban
• Format: Article
• Language: English
• Subjects: 14-3-3 Proteins - chemistry; 14-3-3 Proteins - genetics; Affinity; Amino Acid Sequence; Atomic structure; Binding; Binding Sites; Biological activity; Biophysics; Calorimetry; Cancer; Circular dichroism; Cloning, Molecular; Dichroism; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorescence; Gene Expression; Humans; Kinases; Kinetics; Ligands; Magnetic resonance; MEK kinase; Molecular chains; Molecular Docking Simulation; Molecular modelling; Molecular Sequence Data; NMR; Nuclear magnetic resonance; Peptides; Peptides - chemical synthesis; Peptides - chemistry; Phosphorylation; Pliability; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Proto-Oncogene Proteins c-raf - chemistry; Proto-Oncogene Proteins c-raf - genetics; Raf protein; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Serine; Spectroscopy; Thermodynamics; Titration; Titration calorimetry
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0135976

Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615-630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution.