Project information
Detailed binding scheme and structural determination of the 14-3-3? in complex with a double phosphorylated human tyrosine hydroxylase 1 (MODULATOR)

Information

This project doesn't include Faculty of Science. It includes Central European Institute of Technology. Official project website can be found on muni.cz.
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Project Identification
618460
Project Period
10/2013 - 4/2014
Investor / Pogramme / Project type
European Union
MU Faculty or unit
Central European Institute of Technology

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 300 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.

I propose to apply the methyl-transverse relaxation optimized NMR spectroscopy on deuterated 14-3-3ζ protein with protonated methyl groups of Val, Leu and Ile. Twenty six exposed side-chains of Val, Leu and Ile, located on the inner surface of 14-3-3ζ, will serve as reference points for the intramolecular NOEs between double-phosphorylated human tyrosine hydroxylase 1 (dp_hTH1) and 14-3-3ζ dimer. This approach will be combined with restrained molecular dynamics simulation for phosphorylated residues and 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. A detailed scheme of binding between dp_hTH1 and 14-3-3ζ will be determined based on chemical changes of selectively labeled methyl groups, dp_hTH1’s phosphorous groups, and their neighboring regions.

The proposed approach will have general applicability to most doubly phosphorylated 14-3-3 protein ligands. The research proposed here will not only deepen our understanding of 14-3-3 function but will also enhance our knowledge of essential basic mechanisms with respect to key regulatory proteins.

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