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.

• MeSH
• X-Ray Diffraction MeSH
• Phosphoproteins antagonists & inhibitors   chemistry   MeSH
• Phosphorylation MeSH
• Rats MeSH
• Scattering, Small Angle MeSH
• Eye Proteins antagonists & inhibitors   chemistry   MeSH
• Protein Domains MeSH
• 14-3-3 Proteins chemistry   metabolism   MeSH
• GTP-Binding Protein Regulators antagonists & inhibitors   chemistry   MeSH
• Proteolysis MeSH
• Proton Magnetic Resonance Spectroscopy MeSH
• Protein Structure, Secondary MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphoproteins MeSH
• Eye Proteins MeSH
• phosducin MeSH Browser
• 14-3-3 Proteins MeSH
• GTP-Binding Protein Regulators MeSH

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.

• Keywords
• 14-3-3 protein, fluorescence, hydrogen-deuterium exchange, phosducin, protein complex, protein phosphorylation, small-angle x-ray scattering (SAXS),
• MeSH
• Phosphoproteins chemistry   genetics   metabolism   MeSH
• Phosphorylation MeSH
• Rats MeSH
• Humans MeSH
• Models, Molecular MeSH
• Eye Proteins chemistry   genetics   metabolism   MeSH
• 14-3-3 Proteins chemistry   genetics   metabolism   MeSH
• GTP-Binding Protein Regulators chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphoproteins MeSH
• Eye Proteins MeSH
• phosducin MeSH Browser
• 14-3-3 Proteins MeSH
• GTP-Binding Protein Regulators MeSH
• YWHAZ protein, human MeSH Browser

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp(31) being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.

• Keywords
• Analytical Ultracentrifugation, Apoptosis Signal-regulating Kinase 1 (ASK1), Circular Dichroism (CD), Fluorescence, Small-angle X-ray Scattering (SAXS), Thioredoxin,
• MeSH
• Biophysics MeSH
• Circular Dichroism MeSH
• Spectrometry, Fluorescence MeSH
• Protein Conformation MeSH
• MAP Kinase Kinase Kinase 5 metabolism   MeSH
• Oxidation-Reduction MeSH
• Thioredoxins metabolism   MeSH
• Ultracentrifugation MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MAP Kinase Kinase Kinase 5 MeSH
• Thioredoxins MeSH