Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein

• Published in: The Journal of biological chemistry Vol. 290; no. 26; pp. 16246 - 16260
• Main Authors: Kacirova, Miroslava, Kosek, Dalibor, Kadek, Alan, Man, Petr, Vecer, Jaroslav, Herman, Petr, Obsilova, Veronika, Obsil, Tomas
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.06.2015; American Society for Biochemistry and Molecular Biology
• Subjects: 14-3-3 protein; 14-3-3 Proteins - chemistry; 14-3-3 Proteins - genetics; 14-3-3 Proteins - metabolism; Amino Acid Sequence; Animals; Binding Sites; Eye Proteins - chemistry; Eye Proteins - genetics; Eye Proteins - metabolism; fluorescence; GTP-Binding Protein Regulators - chemistry; GTP-Binding Protein Regulators - genetics; GTP-Binding Protein Regulators - metabolism; Humans; hydrogen-deuterium exchange; Models, Molecular; phosducin; Phosphoproteins - chemistry; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation; Protein Binding; protein complex; protein phosphorylation; Protein Structure and Folding; Protein Structure, Tertiary; Rats; small-angle x-ray scattering (SAXS); hydrogen-deuterium exchange; small-angle x-ray scattering (SAXS); 14-3-3 protein; fluorescence; phosducin; protein phosphorylation; protein complex
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.636563

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. Background: Phosducin is a conserved regulatory phosphoprotein involved in phototransduction whose function is regulated in a 14-3-3-dependent manner. Results: The 14-3-3 protein binding affects the structure and the accessibility of several regions within both domains of phosphorylated phosducin. Conclusion: The 14-3-3 protein sterically occludes the whole Gtβγ binding interface of phosducin. Significance: Mechanistic explanation is given for the 14-3-3-dependent inhibition of phosducin function.