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 ??-subunits of G protein transducin (Gt??). The ability of Pdc to bind to Gt?? 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 Gt?? 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 Gt?? 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 Gt?? 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 Gt?? is based on steric occlusion of Pdc's Gt?? binding surface.

SUBMITTER: Kacirova M 

PROVIDER: S-EPMC5390052 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature