title: Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein.
abstract: Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.
authors.affiliation: From the Departments of Physical and Macromolecular Chemistry and the Institutes of Physiology and.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Kacirova
authors.affiliation: From the Departments of Physical and Macromolecular Chemistry and the Institutes of Physiology and.
authors.forename: Dalibor
authors.initials: D
authors.lastname: Kosek
authors.affiliation: Microbiology,Czech Academy of Sciences, 14220 Prague, and Biochemistry Faculty of Science, Charles University in Prague, 12843 Prague.
authors.forename: Alan
authors.initials: A
authors.lastname: Kadek
authors.affiliation: Microbiology,Czech Academy of Sciences, 14220 Prague, and Biochemistry Faculty of Science, Charles University in Prague, 12843 Prague.
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.affiliation: the Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, 12116 Prague, Czech Republic.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Vecer
authors.affiliation: the Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, 12116 Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Herman
authors.affiliation: the Institutes of Physiology and veronika.obsilova@fgu.cas.cz.
authors.forename: Veronika
authors.initials: V
authors.lastname: Obsilova
authors.affiliation: From the Departments of Physical and Macromolecular Chemistry and the Institutes of Physiology and obsil@natur.cuni.cz.
authors.forename: Tomas
authors.initials: T
authors.lastname: Obsil
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D048948:14-3-3 Proteins; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010750:Phosphoproteins; C572235:YWHAZ protein, human; C065631:phosducin
keywords: 14-3-3 protein; fluorescence; hydrogen-deuterium exchange; phosducin; protein complex; protein phosphorylation; small-angle x-ray scattering (SAXS)