In silico structural and thermodynamic studies of the 14-3-3-z complexes

Gabor Nagy, Hana Zigova, Veronika Weisova and Jozef Hritz


CEITEC – Central European Institute of Technology, Masaryk University
 Kamenice 5/ 4A, 62500 Brno, Czech Republic



14-3-3 proteins are important regulatory proteins found in large abundance within the brain. Mammals have seven known isoforms of 14-3-3, and the protein family is vital for the survival of the organism. 14‑3‑3 proteins form functional dimers, and bind to hundreds of phosphorylated   proteins. Associated with multiple diseases [1] such as insulin sensitivity, the Creutzfeldt-Jakob disease and small-cell lung cancer, understanding the principles driving the interactions of 14-3-3 proteins is an important goal that can advance medical research and molecular biology. We study both the intra-molecular interactions of 14-3-3-z dimers, and the interactions with some of their well known protein partners using computational methods and experimental approaches such as molecular dynamics, advanced free-energy calculations and NMR spectroscopy [2].


The interactions between the 14-3-3-z protein and selected phosphopeptides were studied by Hamiltonian Replica Exchange Molecular Dynamics (H-REMD), and potential-of-mean-force (PMF) methods allowing the calculation of binding affinities. Combined with a novel reaction coordinate approach (distancefield) that was recently proposed [3], these methods allow for the extensive sampling of the binding/unbinding pathways. The calculated binding affinities are compared with the available experimental data.


Comparison of the behavior of selected 14-3-3-z variants in both the monomeric and dimeric states was performed by set of conventional Molecular Dynamics (MD) simulations. The impact of selected mutations at the N-terminal interface on the dimer stability was investigated. Structural changes induced by the C-terminal tail and phosphopeptide binding in different quarternary structure arrangements of the  protein were analysed in detail.


The combination of these methods, provide deeper insight into the molecular processes affecting the behavior of 14-3-3 proteins, and allow for a more accurate prediction of binding affinities towards biologically relevant interaction partners.


1.   Molzan M.,  Ottmann C., Synergistic Binding of the Phosphorylated S233- and S259-Binding Sites of C-RAF to One 14-3-3ζ Dimer, J. Mol. Biol. (2012) 423, 486–495.

2.   Hritz J.; Byeon I-J.; Krzysiak T.; Martinez A.; Sklenář V.; Gronenborn A.M. Dissection of binding between a phosphorylated tyrosine hydroxylase peptide and 14-3-3ζ: a complex story elucidated by NMR. Biophys. J. 2014, 107, 2185-2194.

3.   de Ruiter A., Oostenbrink C., Protein -ligand binding from distancefiled distances  and  Hamiltonian replica exchange simulations, J.Chem.Theory Comput. 2013, 9, 883−892.


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/ 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.