The study of structure and function of 14-3-3 proteins and their complexes
Our research team has been studying the 14-3-3 proteins which are highly conserved regulatory molecules found in all eukaryotes. First they have been isolated from the bovine brain and their unusual name 14-3-3, originates from their elution and migration pattern on two-dimensional DEAE-cellulose chromatography and starch gel electrophoresis. 14-3-3 proteins have the ability of binding the functionally different signal proteins, including kinases, fosfatases and transmembrane receptors by changing their function. Through the functional modulation of a wide range of binding partners, 14-3-3 proteins are involved in many processes, including cell cycle regulation, metabolism control, apoptosis, and control of gene transcription. More than 300 proteins have been described as binding partners till now.
Main goal of these projects is mechanistic understanding of the 14-3-3 protein function in the regulation of selected 14-3-3 binding partners: for example the interaction of 14-3-3 protein with forkhead transcription factor FOXO4, with the regulatory domain of the tyrosine hydroxylase, interaction with ASK1 kinase, with the regulator of G-protein signaling RGS3 and phosducin. Recently, we have been studying two yeast isoforms of 14-3-3 protein (Bmh1 and Bmh2) and the interaction of Bmh with neutral trehalase (Nth1) in yeast.
Methods
- Biochemical approach (recombinant protein expression, site-directed mutagenesis, enzyme kinetics)
- Biophysical approach (fluorescence spectroscopy, analytical ultracentrifugation, isotermal titration calorimetry, surface plasmon resonance, H/D exchange, mass spectrometry, protein structure modeling, X-ray crystallography, molecular dynamics simulations).
- Crystallography of selected complexes
→ These methods enable us to better understand the details how the activity and function of protein-protein complexes is regulated.
Gallery
HD-MS reveals the binding surface of pNth1. Graphs represent HDX kinetics for selected pNth1 regions that show slower exchange kinetics upon Bmh1 binding. A. Regions from unstructured N-terminal part. B. Regions from the catalytic trehalase domain mapped on its homology structural model.
Macakova, Kopecka et. al., BBA-Gen. Subjects 2013, 1830:4491-4499.
Specific trehalase activity of pNth1 WT and mutants upon the Bmh1 activation in vitro
Veisova et. al., Biochemical Journal 2012, 443:663-670.
Structure of human 14-3-3 protein (zeta isoform) with the modeled C-terminal segment
Veisova et. al., Biochemistry 2010, 49(18):3853-61.
Continuous distribution of sedimentation coefficients, c(s), for Bmh1 and Nth1 alone and in the complex
Veisova et. al., Biochemical Journal 2012, 443:663-670.
Solved structures
DNA-binding domain of forkhead transcription factor FOXO4 bound to the DNA
Boura et. al., Acta Crystallographica D 2010, 66, 1351-7
RGS domain of RGS3 (regulator of G-protein signaling)
Rezabkova et. al., Journal of Structural Biology 2010, 170, 451-461.
The study of the cytoplasmatic domains of TRP channels.
Transient receptor potential (TRP) channels are a wide family of non-selective ion channels responsible for monovalent and divalent cation influx into the cells. Members of this family are involved in many sensory processes such as invertebrate vision and hearing, mammalian temperature-, mechano- and chemo-sensation. The TRP channels discovered so far can be divided into seven subfamilies according to their primary structure: TRPV, TRPC, TRPA, TRPM, TRPP, TRPML and TRPN. All are predicted to have six transmembrane helices (S1–S6) and a pore-forming loop between S5 and S6, with varying sizes of intracellular amino and carboxy termini, and are thought to form tetrameric assemblies. Both the N- and C-terminal intracellular domains are comprised of many different domains that are responsible for binding different compounds that can regulate the channels. Our goal is to provide the structural insight into the interactions of TRP channels with ATP, calmodulin and PIP.
