Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and the modulation of blood pressure, is controlled in the phosphorylation-dependent manner including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown [1-3]. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle X-ray scattering, circular dichroism, quenching of tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange coupled to mass spectrometry and nuclear magnetic resonance. 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-depedent inhibition of Pdc function. 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 intrinsically disordered protein which remains likely highly flexible when bound to 14-3-3 indicating the fuzzy-like character of this complex. In addition, it has been speculated that the 14-3-3 protein binding decreases the rate of Pdc dephosphorylation after a light stimulus by virtue of its interaction with phosphorylated Ser54 and Ser73, thus lengthening the time that Pdc remains phosphorylated after a light exposure. Pdc is dephosphorylated in vivo by protein phosphatases 1 (PP1) and 2A (PP2A). Indeed, our dephosphorylation experiments with PP1 revealed that the 14-3-3 protein does slow down the dephosphorylation of doubly phosphorylated Pdc in vitro.
This work was supported by the Czech Science Foundation (Project P305/11/0708), Grant Agency of Charles University in Prague (Project 793913); and Academy of Sciences of the Czech Republic (Research Projects RVO: 67985823 of the Institute of Physiology).