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Detailed binding scheme and structural determination of the 14-3-3ζ in complex with a double phosphorylated human tyrosine hydroxylase 1

Objective

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 human tyrosine hydroxylase 1 (hTH1) regulatory domain in apo form and in the complex with 14-3-3 ζ 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 14-3-3ζ (~75kDa) will be solved by applying of the methyl-transverse relaxation optimized NMR spectroscopy on a deuterated 14-3-3ζ protein with protonated methyl groups of Val, Leu and Ile. Exposed side-chains of 26 Val, Leu and Ile residues will serve as reference points for the intramolecular NOEs between a double-phosphorylated hTH1 (dp_hTH1) and 14-3-3ζ 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 14-3-3ζ will be determined.

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 also enhance our knowledge of essential basic mechanisms with respect to key regulatory proteins.

Field of science

  • /natural sciences/chemical sciences/analytical chemistry/spectroscopy
  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins

Call for proposal

FP7-PEOPLE-2013-CIG
See other projects for this call

Funding Scheme

MC-CIG - Support for training and career development of researcher (CIG)

Coordinator

Masarykova univerzita
Address
Zerotinovo Namesti 9
60177 Brno Stred
Czechia
Activity type
Higher or Secondary Education Establishments
EU contribution
€ 100 000
Administrative Contact
Veronika Papouskova (Dr.)