Laboratory of Structural Biology of Signaling Proteins

Prize of Otto Wichterle for Dr. Dalibor Košek.

Dalibor Kosek received the prize of Otto Wichterle for excellent young scientists of CAS in the year 2022.

In this structural study, the researchers performed a detailed characterization of the interactions in the FOXO4: p53 complex using an integrated approach involving analytical ultracentrifugation, nuclear magnetic resonance, and chemical cross-linking coupled to mass spectrometry. Because both FOXO4 and p53 have multiple domains (see Figure), they studied the role of individual domains and disordered segments of both proteins and mapped their interaction interfaces. They found out that the interaction between p53 transactivation domain TAD and the FOXO4 Forkhead domain is crucial for the overall stability of the p53:FOXO4 complex. Furthermore, contacts involving the N-terminal disordered FOXO4 segment, the C-terminal negative regulatory domain of p53, and the DNA-binding domains of both proteins stabilize the complex formation. By measuring DNA binding, they further found that the p53: FOXO4 complex formation blocks p53 binding to DNA without affecting the DNA-binding properties of FOXO4.

Left, sedimentation velocity analytical ultracentrifugation analysis of interaction between FOXO4 and p53. Middle, chemical shift perturbations obtained from 1H-15N HSQC spectra of 15N-labeled FOXO4 in the presence of p53 mapped onto the crystal structure of the FOXO4 DBD:DNA complex. Right, fluorescence anisotropy measurements showing that the complex formation reduces the DNA-binding affinity of p53.

Mandal R, Kohoutova K, Petrvalska O, Horvath M, Srb P, Veverka V, Obsilova V and Obsil T. FOXO4 interacts with p53 TAD and CRD and inhibits its binding to DNA. Protein Sci. roč. 31, č. 5 (2022), č. článku e4287. IF = 6.725. DOI: 10.1002/pro.4287.

In order to inhibit the FOXO3 activity, it was first necessary to identify small molecule compounds that could block the interaction between FOXO3 and DNA. Using the structural data of FOXO3 DBD and FOXO4 DBD, the researchers developed six different pharmacophore models that were used for in silico screening of small molecule compound databases. Selected compounds were then tested for their ability to inhibit FOXO3 function both in vitro, and in cancer cell lines. The interactions of these compounds with FOXO3 DBD were assessed using NMR spectroscopy and docking studies.

The teams of Dr. V. Obšilová from the Institute of Physiology CAS in BIOCEV and prof. T. Obšil from the Faculty of Science, Charles University with the team of prof. M. J. Ausserlechner from the Medical University of Innsbruck, Austria identified the compounds S9 and its oxalate salt S9OX as compounds able to inhibit the FOXO3 activity in cancer cells. They also proposed that due to their mode of binding to FOXO3 DBD, these compounds may also interfere with protein-protein interactions of FOXO3. The advantage of these compounds is the strict control of application-dose and -time and the fact that they are not immunogenic allowing repeated applications – so dose- and application time can be adjusted to damage cancer cells or boost anti-cancer immunity, but also limit unwanted side effects of FOXO-inhibition on stem cells and other somatic tissues. Future research will be focused on investigation whether or not S9 can be used as a chemical foundation for developing FOXO-regulatory compounds that would control the functions and target gene subsets of FOXO transcription factors.

Original paper: Hagenbuchner J^*, Obsilova V^*, Kaserer T^*, Kaiser N, Rass B, Psenakova K, Docekal V, Alblova M, Kohoutova K, Schuster D, Aneichyk T, Vesely J, Obexer P, Obsil T⁺, Ausserlechner MJ⁺. Modulating FOXO3 transcriptional activity by small, DBD-binding molecules. eLife. 2019, 8(Dec 4), e48876. doi: 10.7554/eLife.48876. IF = 7.551

Compounds S9 blocks the DNA binding surface of Forkhead transcription factor FOXO3. The figure shows the structural model of the DNA-binding domain of FOXO3 with bound compound S9 based on data from NMR measurements and docking simulations.

We obtained the first prize for the best publication in Physiological Research with the young authors from for the year 2018.

We got the prize for the best publication of the Institute of Physiology for year 2017 for the publication:

Alblova M, Smidova A, Docekal V, Vesely J, Herman P, Obsilova V, Obsil T. :

Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1.

Proc Natl Acad Sci U S A. (2017) 114:E9811-E9820. IF = 9.504.

Alblova M, Smidova A, Docekal V, Vesely J, Herman P, Obsilova V, Obsil T.
Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1.

Proc Natl Acad Sci U S A. 2017 Oct 30. pii: 201714491. doi: 10.1073/pnas.1714491114.
https://www.ncbi.nlm.nih.gov/pubmed/29087344

The 14-3-3 proteins, a family of highly conserved scaffolding proteins ubiquitously expressed in all eukaryotic cells, interact with and regulate the function of several hundreds of partner proteins. Yeast neutral trehalases (Nth), enzymes responsible for the hydrolysis of trehalose to glucose, compared with trehalases from other organisms, possess distinct structure and regulation involving phosphorylation at multiple sites followed by binding to the 14-3-3 protein. Here we report the crystal structures of yeast Nth1 and its complex with Bmh1 (yeast 14-3-3 isoform), which, together with mutational and fluorescence studies, indicate that the binding of 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. The presented structure of the Bmh1:Nth1 complex highlights the ability of 14-3-3 to modulate the structure of a multidomain binding partner and to function as an allosteric effector. Furthermore, comparison of the Bmh1:Nth1 complex structure with those of 14-3-3:serotonin N-acetyltransferase and 14-3-3:heat shock protein beta-6 complexes revealed similarities in the 3D structures of bound partner proteins, suggesting the highly conserved nature of 14-3-3 affects the structures of many client proteins.

Our new paper in JBC: August 2016 Structural Insight into the 14-3-3 Protein-Dependent Inhibition of Protein Kinase ASK1.

Petrvalska O, Kosek D, Kukacka Z, Tosner Z, Man P, Vecer J, Herman P, Obsilova V and Obsil T. J. Biol. Chem. jbc.M116.724310.

First Published on August 11, 2016, doi:10.1074/jbc.M116.724310

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated S966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study, we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at S966 (pASK1-CD) and the 14-3-3ζ protein. Small Angle X-ray Scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD:14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.

Kopecka M, Kosek D, Kukacka Z, Rezabkova L, Man P, Novak P, Obsil T, Obsilova V.

J Biol Chem. 2014 May 16;289(20):13948-61.

http://www.ncbi.nlm.nih.gov/pubmed/24713696

Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.

Kacirova M, Kosek D, Kadek A, Man P, Vecer J, Herman P, Obsilova V and Obsil T

J. Biol. Chem. jbc.M115.636563. First Published on May 13, 2015, doi:10.1074/jbc.M115.636563

Kacirova M, Kosek D, Kadek A, Man P, Vecer J, Herman P, Obsilova V and Obsil T

http://www.ncbi.nlm.nih.gov/pubmed/25971962

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in the phosphorylation-dependent manner including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle X-ray scattering, time-resolved fluorescence spectroscopy and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3 binding motifs are located, is intrinsically disordered protein which reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.

Prof. RNDr. Tomáš Obšil, Ph.D. was awarded by the title of professor at The Faculty of Science, Charles University, majoring in Physical Chemistry. Professors graduation will take place December 18, 2014.

16. 12. 2014: The Foundation of Josef, Marie and Zdeňek Hlávka awarded Hana Janoušková for being the best graduated student from 2nd Medical Faculty, Charles University in Prague.

Kosek D, Kylarova S, Psenakova K, Rezabkova L, Herman P, Vecer J, Obsilova V, Obsil T.

J Biol Chem. 2014 Aug 29;289(35):24463-74.

http://www.ncbi.nlm.nih.gov/pubmed/25037217

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp(31) being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.

14-10061S – Mechanismus regulace kinasové aktivity proteinkinasy ASK1 (řešitelé: T. Obšil, PřF UK, a V. Obšilová, FgÚ AV ČR, v.v.i.)

21. 2. 2014 – Assoc. Prof. RNDr. Tomáš Obšil awarded by students of the Faculty of Science of Charles University as the best teacher in the field of chemistry

Assoc. Prof. Tomáš Obšil received the Studentský Velemlok award for the best teacher in 2013 from the Dean of the Faculty of Science of Charles University in Prague on 21. 2014. Teachers are nominated for this award by students based on the results of the evaluation of their teaching in a student survey.