We study molecular mechanisms by which protein function can be regulated. In particular, we are interested in 14-3-3 proteins and their complexes with proteins involved in apoptosis, cancer, G-protein and calcium-triggered signaling pathways. We employ both biophysical (fluorescence spectroscopy, analytical ultracentrifugation, SAXS, mass spectrometry, X-ray crystallography, NMR, protein structure modeling, etc.) and biochemical (recombinant protein expression, site-directed mutagenesis) approaches to understand the details of how the activity and function of protein-protein complexes are regulated.
14-3-3 proteins · Phosducin (Pdc) · Neutral trehalase Nth1 · Protein kinase ASK1 · FOXO-DBD · Protein kinase CaMKK2 · Stabilization of protein-protein interactions · Solved structures
14-3-3 proteins are a family of regulatory molecules, which specifically bind to phosphoserine (or phosphothreonine)- containing motifs (pSer/pThr) in a sequence-specific manner. Through these binding interactions, 14-3-3 proteins play key regulatory roles in signal transduction, cell cycle control, metabolism control and apoptosis. More than 200 14-3-3 binding partners have been reported so far and some of them play prominent roles in cancer development (e.g. transcription factors p53 and FOXO), neurodegeneration (e.g. Tau protein, ASK1 kinase), cardiovascular diseases (e.g. RGS proteins, phosducin) or inflammation (e.g. NFkB, ASK1 kinase). Mechanistically, 14-3-3 proteins act as allosteric regulators and/or molecular scaffolds that constrain the conformation of the binding partner; if the target protein is an enzyme, this can affect its catalytic activity. Nonetheless, the underlying molecular mechanisms are only partially identified, mainly due to the lack of structural data.
Structure of the 14-3-3 dimer
Crystal structure of the 14-3-3 protein (human zeta isoform, PDB ID code 1QJB [Rittinger et al. (1999) Mol. Cell 4, 153]). (Top) The ribbon representation. (Bottom) The surface representation. Residues that are totally conserved among all seven human isoforms are shaded in dark red.
Modes of 14-3-3 protein action
The 14-3-3 protein binding can: (i) induce a conformational change of the target protein; (ii) mask a specific region in the target protein; or (iii) facilitate the interaction between two other proteins.
Phosducin (Pdc) is a highly conserved acidic phosphoprotein that regulates visual signal transduction by modulating the
amount of transducin Gtαβγ heterotrimer through competition with the Gta subunit for binding to the Gtβγ complex. Pdc was also shown to regulate the cardiovascular system by modulating sympathetic activity and blood pressure. Phosducin function is regulated through the phosphorylation and the binding to the 14-3-3 protein. Main goal of this project was to elucidate solution structure of the 14-3-3:pPdc complex and provide a mechanistic explanation for the inhibitory effect of the 14-3-3 protein binding on interaction between phosducin and G protein. This project was funded by Czech Science Foundation (Project P305/11/0708).
Results were published in: Rezabkova et al. (2012) Biophys. J.; Kacirova et al. (2015) J. Biol. Chem.; Kacirova et al. (2017) Biophys. J.
Solution structure of the 14-3-3:pPdc complex
The best-scoring model of the pPdc:14-3-3ζ complex calculated using the AllosMod-FoXS simulation against the SAXS data (Kacirova et al. (2017) Biophys. J.). Residues directly involved in Gtβγ binding are shown in green.
Neutral trehalase Nth1
Neutral trehalase 1 (Nth1) catalyses the hydrolysis of trehalose to glucose. Trehalose is found in many organisms ranging from bacteria to higher plants, and in yeast it functions as a major storage carbohydrate, carbon source and a stress protectant. The activity of yeast Nth1 is triggered by the protein 14-3-3, an interaction that is phosphorylation dependent. Main goal of this project is to elucidate the molecular mechanism of the 14-3-3-dependent activation of yeast Nth1. This project was funded by Czech Science Foundation (Project P207/11/0455).
Results were published in: Veisova et al. (2012) Biochem. J.; Macakova et al. (2013) Biochim. Biophys. Acta; Kopecka et al. (2014) J. Biol. Chem.; Kopecka et al. (2017) PNAS USA.
Crystal structure of the complex between phosphorylated Nth and Bmh1 (yeast 14-3-3 protein) (PDB 5N6N). The protomers of the Bmh1 homodimer are shown in yellow and brown. Nth1 is shown in blue. The phosphorylated Ser60 and Ser83 are shown as sticks. The calcium ion is shown in orange.
Structural analysis revealed that the binding of phosphorylated Nth1 by 14-3-3 triggers Nth1’s activity by enabling the proper 3D configuration of Nth1’s catalytic and calcium-binding domains relative to each other, thus stabilizing the flexible part of the active site required for catalysis (Alblova et al. (2017) PNAS USA).
Protein kinase ASK1
Protein kinase ASK1, a member of the mitogen-activated protein kinase kinase kinase family, activates JNK and p38 MAP kinase signaling pathways in response to various stress stimuli, including oxidative stress, endoplasmic reticulum stress, and calcium ion influx . ASK1 plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration and cardiovascular diseases and is considered as a promising therapeutic target. The activity of ASK1 is regulated by several other proteins including thioredoxin and the 14-3-3 protein that both function as physiological inhibitors of ASK1. Main goal of this project is to elucidtae the inhibitory mechanism of ASK1 through interaction with regulatory proteins 14-3-3 and thioredoxin (TRX1). This project was funded by Czech Science Foundation (Project 14-10061S).
Results were published in: Kosek et al. (2014) J. Biol. Chem.; Petrvalska et al. (2016) J. Biol. Chem.; Kylarova et al. (2016) FEBS J.
The activation mechanism of ASK1 in response to oxidative stress. ASK1 forms homo-oligomer thorough the C-terminal coiled-coil domain. In steady state, Trx and 14-3-3 proteins bind to ASK1 and negatively regulate the activation of ASK1. In response to oxidative stress, Trx and 14-3-3 proteins dissociate from ASK1 while TRAF2 and TRAF6 are recruited to ASK1 and bind to ASK1, resulting in homophilic interaction of ASK1 through the N-terminal coiled-coil domain and activation of ASK1. Adapted from Shiizaki et al. Advances in Biological Regulation (2012).
FOXO transcription factors control apoptosis, stress resistance and longevity in mammalian cells. Although the members of the FOXO family act as tumor suppressors in some cell types, emerging evidence suggests that FOXO3 also contributes to tumor stem cell renewal, immune suppression, metastases and chemotherapy resistance in certain cancer types. By a combined in silico / cell biological screening approach several small, drug-like compounds that interact with the DNA-binding domain of FOXO3 and efficiently inhibit its transcriptional activity have been identified. The main aim of this project is to define the structural basis for the interaction between compounds and FOXO3 by NMR spectroscopy, design and develop derivatives with improved properties regarding solubility and affinity, analyze compound-dependent inhibition of target recognition by FOXO3 in vitro and in vivo, study the effects of compounds on FOXO3-induced cancer cell survival in 3D cell culture and in vivo.
This project is a collaboration with the group of prof. Michael J. Ausserlechner from Medical University Innsbruck, Innsbruck, Austria, and it is funded by Czech Science Foundation (Project 17-33854L).
Crystal structure of the FOXO-DNA-binding domain bound to DNA (PDB 3L2C):
Protein kinase CaMKK2
The Ca2+/calmodulin-dependent protein kinase (CaMK) cascade is involved in the regulation of many physiological and pathophysiological processes. This signaling cascade consists of CaMKI and CaMKIV and their upstream activator CaMK kinase (CaMKK). The activity of CaMKK is inhibited through phosphorylation by PKA in a process involving the binding to the 14-3-3 protein. However, the molecular mechanism of this 14-3-3-mediated inhibition of CaMKK is currently unknown. Anticipated mechanisms include direct inhibition through structural modulation of the catalytic site, blocking of dephosphorylation of inhibitory phosphorylation sites or interference with the binding of Ca2+/calmodulin to CaMKK. Main goal of this project is to elucidate the molecular basis of this regulation by performing functional and structural analysis of interactions between 14-3-3 and CaMKK2. This project is funded by Czech Science Foundation (Project 16-02739S).
Stabilization of protein-protein interactions
14-3-3 proteins regulate function of more than 200 binding partners including proteins playing prominent roles in cancer development (e.g. transcription factors p53 and FOXO), neurodegeneration (e.g. Tau protein, ASK1 kinase), cardiovascular diseases (e.g. RGS proteins, phosducin) or inflammation (e.g. NFkB, ASK1 kinase). Thus specific modulation of these protein-protein interactions (e.g. between 14-3-3 and FOXO or ASK1) by small-molecule compounds can provide promissing drug candidates as well as to help us to understand the 14-3-3 signalling biology. The project is done in collaboration with the group of prof. Christian Ottmann from Technische Universiteit Eindhoven and other partners and it is funded by H2020-MSCA-ITN project 675179 TASPPI (http://www.tasppi.eu/about/).
List of crystal structures solved by our group:
Smidova et al. (2018) FEBS J.
Interactions between 14-3-3 and the 14-3-3-binding motifs of caspase-2. (A) Crystal structure of the 14-3-3gamma:pepS139 complex. The 2Fo-Fc electron density map is contoured at 1σ. (B) Detailed view of contacts between 14-3-3c and the pepS139 peptide. The caspase-2 residues are labeled in red, and the 14-3-3c residues are labeled in black. (C) Crystal structure of the 14-3-3c:pepS164 complex. The 2Fo-Fc electron density map is contoured at 1σ. (D) Detailed view of contacts between 14-3-3gamma and the pepS164 peptide. The caspase-2 residues re labeled in red, and the 14-3-3c residues are labeled in black.
Psenakova et al. (2018) Biochim Biophys Acta Gen Subj.
Contacts between 14-3-3 and the 14-3-3 binding motifs of phosphorylated CaMKK2. (A) Crystal structure of the 14-3-3ζ:pepS100 complex. The 2Fo-Fc electron density map is contoured at 1σ. (B) Detailed view of contacts between 14-3-3ζ and the pepS100 peptide. The CaMKK2 residues are labeled in red, and the 14-3-3ζ residues are labeled in black. (C) Crystal structure of the 14-3-3γ:pepS511 complex. The 2Fo-Fc electron density map is contoured at 1σ. (D) Detailed view of contacts between 14-3-3γ and the pepS511 peptide. The CaMKK2 residues are labeled in red, and the 14-3-3γ residues are labeled in black.
PDB ID: 5N6N
Alblova et al. (2017) Proc. Natl. Acad. Sci. U.S.A.
PDB ID: 5JTA
Alblova et al. (2017) Proc. Natl. Acad. Sci. U.S.A.
PDB ID: 5M4A
Alblova et al. (2017) Proc. Natl. Acad. Sci. U.S.A.
PDB ID: 6EJL
PDB ID: 3L2C
Boura et al. (2010) Acta Crystallogr.,Sect.D
PDB ID: 2OJ4
Rezabkova et al. (2010) J.Struct.Biol.