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.
Our recent 31P NMR data showed much more complex binding mode between 14-3-3z and doubly phosphorylated peptide of human tyrosine hydroxylase 1 (hTh1) than was originally thought [1]. Analysis of the binding data revealed that the 14-3-3ζ dimer and the S19- and S40-doubly phosphorylated hTh1 peptide interact in multiple ways, with three major complexes formed: (1) a single peptide bound to a 14-3-3ζ dimer via the S19 phosphate with the S40 phosphate occupying the other binding site; (2) a single peptide bound to a 14-3-3ζ dimer via the S19 phosphorous with the S40 free in solution; or (3) a 14-3-3ζ dimer with two peptides bound via the S19 phosphorous to each binding site.
Experimental determination of the binding affinities and binding modes between 14-3-3ζ dimer and their phosphorylated protein partners is very tedious therefore we have decided to address this problem also by computational techniques. Binding/unbinding pathways and the corresponding absolute binding affinities of the selected phosphopeptides with respect to the 14-3-3ζ have been studied by Hamiltonian Replica Exchange Molecular Dynamics (H-REMD) combined with a novel reaction coordinate approach (distancefield), and potential-of-mean-force (PMF) methods [2]. Our preliminary computational results are compared with the available experimental data.
The combination of the advanced NMR and computational approaches deepened our understanding about the binding mechanism of 14-3-3 proteins with their phosphorylated protein partners. The presented approaches have general applicability for almost any 14-3-3/phophopeptide compex.
The project is financed from the SoMoPro II programme. The research leading to this invention has acquired a financial grant from the People Programme (Marie Curie action) of the Seventh Framework Programme of EU according to the REA Grant Agreement No. 291782. The research is further co-financed by the South-Moravian Region. The article/paper reflects only the author´s views and the Union is not liable for any use that may be made of the information contained therein. In addition, this work was also supported by Czech Science Foundation (I 1999-N28) and the project “SYLICA - Synergies of Life and Material Sciences to Create a New Future” (286154). This work was realized in CEITEC – Central European Institute of Technology with research infrastructure supported by the project CZ.1.05/1.1.0.0/02.0068 financed from European Regional Development Fund. The computational simulations were realized in the National Supercomputing Center IT4Innovations, which is supported by the Op VaVpI project number CZ.1.05/1.1.00/02.0070. Further Computational resources were provided by the MetaCentrum under the program LM2010005 and the CERIT-SC under the program Centre CERIT Scientific Cloud, part of the Operational Program Research and Development for Innovations, Reg. no. CZ.1.05/3.2.00/08.0144.