MODULATOR · Detailed binding scheme and structural determination of the 14-3-3ζ in complex with a double phosphorylated human tyrosine hydroxylase 1
FP7 — People (Marie Curie Actions)
- Duration
- 2013-10-01 → 2017-09-30
- EU contribution
- €100,000
- Participants
- 1
- Scheme
- MC-CIG
Lines connect the coordinator with its partners.
Results in brief
Detailed binding scheme and structural determination of the 14-3-3ζ in complex with a double phosphorylated human tyrosine hydroxylase 1
This early termination report covers 7 months period since 1st October 2013 - 30th April 2014. Members of the 14-3-3 protein family are important modulators of several key signaling pathways that regulate critical biological activities, including cell cycle control, cell growth, proliferation, and apoptosis. Human tyrosine hydroxylase 1 (hTH1) activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator protein 14-3-3zeta. Human 14-3-3zeta isoforms form very stable homo-dimer. In order to study the effect of monomerization of 14-3-3zeta protein, mutations at the dimer interface were designed and incorporated into the 14-3-zeta gene. Purity of the 14-3-3zeta mutants and wild type was checked by SDS-PAGE electrophoresis and MALDI mass spectrometry. NATIVE PAGE electrophoresis revealed that one of two tested 14-3-3zeta mutants exists almost exclusively in the monomeric state. Other two high purity samples prepared within the time frame of the project were C-tail truncated construct of 14-3-3zeta and doubly phosphorylated peptide (1-50 region) of hTH1 (dp-hTH1-50). All samples will be further analyzed and used for detailed study of 14-3-3zeta protein monomerization. The project web site: http://www.ceitec.eu/project-modulator/t1816
Data: CORDIS, © European Union
Project 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.
Original text from CORDIS.
Participants
- Masarykova univerzita · BrnoCoordinatorCzechia
Links
Data: CORDIS, © European Union