Structural studies on Pdc/14-3-3 protein complex

Miroslava Kacirova^1,2, Jiri Novacek³, Lukas Zidek³ and Tomas Obsil^1,2

¹Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic
²Institute of Physiology, Academy of Sciences of Czech the Republic, 14220 Prague, Czech Rep.
³Masaryk University, CEITEC – Central European Institute of Technology, 60177 Brno, Czech Rep.
kacirova@natur.cuni.cz

Phosducin (Pdc), a highly conserved 30 kDa phosphoprotein, regulates visual signal transduction by interacting with the beta and gamma subunits of the retinal G-protein. Pdc was also suggested to be involved in transcriptional control, the regulation of transmission at the photoreceptor-to-ON-bipolar cell synapse, and the regulation of the sympathetic activity and blood pressure. The function of Pdc is regulated by phosphorylation at Ser54 and Ser73 in a process that involves the binding of phosphorylated Pdc to the regulatory 14-3-3 protein. The 14-3-3 proteins are highly conserved dimeric molecules that regulate the function of other proteins through a number of different mechanisms. The exact role of the 14-3-3 protein in regulating Pdc function is still unclear, but it is entirely possible that 14-3-3 either sterically occludes and/or affects the structure of Pdc. Both 14-3-3 binding motifs are located within the N-terminal domain of Pdc, which participates in the binding to the beta and gamma subunits of the retinal G-protein as well as contains the SUMOylation site Lys-33.

Our previous study revealed that phosphorylated Pdc and the 14-3-3 protein form a stable complex with 1:2 molar stoichiometry. Complex formation with 14-3-3 affects the structure and reduces the flexibility of both the N- and C-terminal domains of dpPdc, suggesting that dpPdc undergoes a conformational change when binding to 14-3-3. To further investigate this interaction and mainly the 14-3-3 protein-mediated conformational changes of Pdc, we performed structural studies using Bio-NMR, SAXS and tryptophan fluorescence whose results are presented here.

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This work was supported by the Czech Science Foundation (Project P305/11/0708), Grant Agency of Charles University in Prague (Projects 28510 and 793913); and Academy of Sciences of the Czech Republic (Research Projects RVO: 67985823 of the Institute of Physiology).