4FJ3

14-3-3 isoform zeta in complex with a diphoyphorylated C-RAF peptide

Summary for 4FJ3

• Entry DOI: 10.2210/pdb4fj3/pdb
• Related: 1A4O 3IQJ 3NKX
• Descriptor: 14-3-3 protein zeta/delta, RAF proto-oncogene serine/threonine-protein kinase (3 entities in total)
• Functional Keywords: 14-3-3 fold, all alpha-helical, adapter protein, protein binding-transferase complex, protein binding/transferase
• Biological source: Homo sapiens (human); More
• Cellular location: Cytoplasm: P63104 P04049
• Total number of polymer chains: 3
• Total formula weight: 57624.64

Authors

Ottmann, C.,Molzan, M. (deposition date: 2012-06-11, release date: 2012-09-05, Last modification date: 2024-11-20)

Primary citation

Molzan, M.,Ottmann, C.
Synergistic binding of the phosphorylated S233- and S259-binding sites of C-RAF to one 14-3-3zeta dimer
J.Mol.Biol., 423:486-495, 2012

PubMed Abstract: C-RAF kinase is a central component of the Ras-RAF-MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase)-ERK (extracellular signal-regulated kinase) pathway, which has been shown to be activated in 30% of human tumors. 14-3-3 proteins inactivate C-RAF by binding to the two N-terminal phosphorylation-dependent binding sites surrounding S233 and S259. 14-3-3 proteins can bind two target sequences located on one polypeptide chain simultaneously, thereby increasing binding affinity compared to single-site binding and possibly allowing regulated 14-3-3 binding through gatekeeper phosphorylation. To date, it was unclear whether 14-3-3 proteins can bind the two N-terminal phosphorylation-dependent binding sites of C-RAF simultaneously. Fluorescence polarization using phosphorylated peptides demonstrated that S233 is the low-affinity and S259 is the high-affinity binding site, while simultaneous engagement of both sites by 14-3-3ζ enhances affinity compared to single-site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3ζ dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3ζ/C-RAFpS233,pS259 complex. Cellular localization studies validate the significance of these sites for cytoplasmic retention of C-RAF, suggesting an extended mechanism of RAF regulation by 14-3-3 proteins.

PubMed: 22922483
DOI: 10.1016/j.jmb.2012.08.009

Experimental method

X-RAY DIFFRACTION (1.95 Å)