title: Structural Basis for the 14-3-3 Protein-Dependent Inhibition of Phosducin Function.
abstract: Phosducin (Pdc) is a conserved phosphoprotein that, when unphosphorylated, binds with high affinity to the complex of βγ-subunits of G protein transducin (Gtβγ). The ability of Pdc to bind to Gtβγ is inhibited through its phosphorylation at S54 and S73 within the N-terminal domain (Pdc-ND) followed by association with the scaffolding protein 14-3-3. However, the molecular basis for the 14-3-3-dependent inhibition of Pdc binding to Gtβγ is unclear. By using small-angle x-ray scattering, high-resolution NMR spectroscopy, and limited proteolysis coupled with mass spectrometry, we show that phosphorylated Pdc and 14-3-3 form a complex in which the Pdc-ND region 45-80, which forms a part of Pdc's Gtβγ binding surface and contains both phosphorylation sites, is restrained within the central channel of the 14-3-3 dimer, with both 14-3-3 binding motifs simultaneously participating in protein association. The N-terminal part of Pdc-ND is likely located outside the central channel of the 14-3-3 dimer, but Pdc residues 20-30, which are also involved in Gtβγ binding, are positioned close to the surface of the 14-3-3 dimer. The C-terminal domain of Pdc is located outside the central channel and its structure is unaffected by the complex formation. These results indicate that the 14-3-3 protein-mediated inhibition of Pdc binding to Gtβγ is based on steric occlusion of Pdc's Gtβγ binding surface.
authors.affiliation: Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic; Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Kacirova
authors.affiliation: CEITEC, Masaryk University, Brno, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Novacek
authors.affiliation: BioCeV-Institute of Microbiology, Czech Academy of Sciences, Vestec, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.affiliation: Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic. Electronic address: veronika.obsilova@fgu.cas.cz.
authors.forename: Veronika
authors.initials: V
authors.lastname: Obsilova
authors.affiliation: Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic; Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic. Electronic address: obsil@natur.cuni.cz.
authors.forename: Tomas
authors.initials: T
authors.lastname: Obsil
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D005136:Eye Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D020555:GTP-Binding Protein Regulators / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D059748:Proteolysis; D066244:Proton Magnetic Resonance Spectroscopy; D051381:Rats; D053838:Scattering, Small Angle; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction
chemical_list: D048948:14-3-3 Proteins; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010750:Phosphoproteins; C065631:phosducin