Informace o projektu
Structural determination of the human 14-3-3zeta in complex with a double phosphorylated human tyrosine hydroxylase 1 (MODULATOR)

Kód projektu
4SGA8679
Období řešení
5/2014 - 12/2016
Investor / Programový rámec / typ projektu
Jihomoravský kraj
Fakulta / Pracoviště MU
Středoevropský technologický institut

14-3-3 proteins, found in all eukaryotic cells, are known to be important in cell-cycle regulation, apoptosis, and regulation of gene expression. They are also associated with oncogenic and neurodegenerative amyloid diseases. 14-3-3 proteins are active as homo- or heterodimers and bind more than 850 diverse target phosphoproteins, thereby forcing conformational changes or/and stabilizing active conformations in their target proteins. To date, no crystal structure is known for a 14-3-3 dimer in complex with a doubly phosphorylated target protein; this prevents a full understanding of the 14-3-3 molecular mechanism.
Spatial structure of hTH1 regulatory domain in apo form and in the complex with 14-3-3zeta will be determined. The structured region of the hTH1 regulatory domain (~10kDa) in apo form will be solved by conventional NMR approach. Much more challenging structure in the complex with 14-3-3zeta (~75kDa) will be solved by applying of the methyl-transverse relaxation optimized NMR spectroscopy on a deuterated 14-3-3zeta protein with protonated methyl groups of Val, Leu and Ile. Exposed side-chains of 26 Val, Leu and Ile residues, located on the inner surface of the 14-3-3zeta, will serve as reference points for the intramolecular NOEs between a double-phosphorylated human tyrosine hydroxylase 1 (dp_hTH1) and 14-3-3zeta dimer. This approach will be combined with the restrained molecular dynamics simulation for phosphorylated residues and a novel Hamiltonian replica exchange, using soft-core interactions developed by myself and Dr. Oostenbrink. The obtained structural ensemble will be refined based on the measured NMR data. Moreover, a detailed scheme of binding between dp_hTH1 and 14-3-3zeta will be determined based on chemical changes of selectively labeled methyl groups, dp_hTH1’s phosphorous groups, and their neighboring regions.

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