14-3-3 C-TERMINAL STRETCH CHANGES ITS CONFORMATION UPON LIGAND BINDING AND PHOSPHORYLATION AT THR232

V. Obšilová², P. Heřman³, J. Večeř³, M. Šulc⁴, J. Teisinger² and T. Obšil^1,2

¹Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague,

²Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague,

³Institute of Physics, Faculty of Mathematics and Physics, Charles University, 121 16 Prague,

⁴Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic

14-3-3 proteins are abundant binding proteins involved in many biologically important processes [1,2]. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3 [3]. Time-resolved fluorescence and molecular dynamics have been used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at T232, a casein kinase I phosphorylation site located within this region. Tryptophan residue placed at position 242 has been exploited as an intrinsic fluorescence probe of the C-terminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3 C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3 at T232 resulted in inhibition of phosphopeptide binding and supression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of W242 also revealed that phosphorylation at T232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamics simulations suggest that phosphorylation at T232 induces more extended conformation of 14-3-3 C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties [4].

This work was supported by Grants 204/03/0714 and 309/02/1479 of the Grant Agency of the Czech Republic; by Grant B5011308 of the Grant Agency of the Czech Academy of Sciences; by Research Projects 1K03020, MSM 1131 00001, and 1132 00001 of the Ministry of Education, Youth and Sports of the Czech Republic, and by Research Project AVOZ 5011922.

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