Structural Basis for the 14-3-3 Protein-Dependent Inhibition of Phosducin Function

0475511 - FGÚ 2018 RIV US eng J - Článek v odborném periodiku
Kacířová, Miroslava - Nováček, J. - Man, Petr - Obšilová, Veronika - Obšil, Tomáš
Structural Basis for the 14-3-3 Protein-Dependent Inhibition of Phosducin Function.
Biophysical Journal. Roč. 112, č. 7 (2017), s. 1339-1349. ISSN 0006-3495. E-ISSN 1542-0086
Grant CEP: GA ČR(CZ) GA16-02739S; GA MŠMT(CZ) LQ1604; GA MŠMT(CZ) ED1.1.00/02.0109
Institucionální podpora: RVO:67985823 ; RVO:61388971
Klíčová slova: phosducin * 14-3-3 protein * NMR spectroscopy * limited proteolysis
Obor OECD: Biochemical research methods; Biochemistry and molecular biology (MBU-M)
Impakt faktor: 3.495, rok: 2017

Phosducin (Pdc) is a conserved phosphoprotein that, when unphosphorylated, binds with high affinity to the complex of beta gamma subunits of G protein transducin. The ability of Pdc to bind to Gt(beta gamma) is inhibited through its phosphorylation at S54 andS73 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(beta gamma) 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 forma complex in which the Pdc-ND region 45-80, which forms a part of Pdc's Gt(beta gamma) 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-NDis likely located outside the central channel of the 14-3-3 dimer, but Pdc residues 20-30, which are also involved in Gt(beta gamma) 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(beta gamma) is based on steric occlusion of Pdc's Gt(beta gamma) binding surface.
Trvalý link: http://hdl.handle.net/11104/0272201