title: Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein.
abstract: 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.
authors: Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata, 700 054, India.
authors: Anirban
authors: A
authors: Ghosh
authors: Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata, 700 054, India.
authors: Bhisma Narayan
authors: BN
authors: Ratha
authors: Department of Molecular Medicine, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata, 700 054, India.
authors: Nilanjan
authors: N
authors: Gayen
authors: Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109-1055, United States of America.
authors: Kamal H
authors: KH
authors: Mroue
authors: Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata, 700 054, India.
authors: Rajiv K
authors: RK
authors: Kar
authors: Department of Molecular Medicine, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata, 700 054, India.
authors: Atin K
authors: AK
authors: Mandal
authors: Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata, 700 054, India.
authors: Anirban
authors: A
authors: Bhunia
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D010766:Phosphorylation; D018583:Pliability; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
chemical_list: D048948:14-3-3 Proteins; D010455:Peptides; D011994:Recombinant Proteins; D019908:Proto-Oncogene Proteins c-raf