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Project MODULATOR

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

ABSTRACT

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.

Figure 1.Crystal structure of 14-3-3ζ in complex with PKCε (pdb: 2WH0).Linker between phosphate sites and flexible C-terminal tails of 14-3-3ζ are not resolved in the crystal structure and, therefore, were modeled by program MODELLER. PKCε peptide is shown in bond representation, 14-3-3ζ in ribbon representation where only surface exposed Val, Leu, Ile are shown explicitly. Site 1 and site 2 for pSer binding are indicated.

PROJECT AIMS

1. Comparison of the binding schemes of the dp_hTH1_50 peptide, dp_hTH1_169 regulatory domain and dp_hTH1_497 full length protein into 14-3-3ζ.
2. Elucidating the impact of C-terminal tail and dimerization of 14-3-3ζ on the binding to dp_hTH1.
3. 3D structural determination of the structured part of hTH1 regulatory domain (70-169).
4. Structure of the 14-3-3ζ in the complex with dp_hTH1_50 and dp_hTH1_169.