Czech National Centre of the European Research Infrastructure Consortium INSTRUCT ERIC

Czech Infrastructure for Integrative Structural Biology – CIISB

Structure without function is a corpse, function without structure is a ghost.

S. Vogel and S. A. Weinwright, 1969

 

A gateway to realm of structural data for biochemists, biophysicists, molecular biologist, and all scientists whose research benefits from accurate structure determination of biological macromolecules, assemblies, and complex molecular machineries at atomic resolution.

Open access to 10 high-end core facilities and assisted expertise in NMR, X-ray crystallography and crystallization, cryo-electron microscopy and tomography, biophysical characterization of biomolecular interaction, nanobiotechnology, proteomics and structural mass spectrometry.

A distributed infrastructure constituted by Core Facilities of CEITEC (Central European Institute of Technology), located in Brno, and BIOCEV (Biotechnology and Biomedicine Centre), located in Vestec near Prague, Central Bohemia.

Research Highlights

the best of science obtained using CIISB Core Facilities

Proc. Natl. Acad. Sci. U.S.A. 2019

Nature Index Journal

(A) Cryo-EM structure of RV-B5 complexed to OBR-5-340 colored radially as indicated by the color bar. Distance from the viral center is 130 Å (white) to 160 Å (dark blue). (B) Example of the quality of the maps of RV-B5 with OBR-5-340 (Left) and without OBR-5-340 (Right). (C) View centered on OBR-5-340 (yellow) in complex with RV-B5 (red). For comparison, the control, i.e., RV-B5 solved in the absence of inhibitor (blue), is overlaid. Residues nearby and contributed by VP1 are labeled. (D) RV-B5 solved in the absence of OBR-5-340. Note the absence of density at the position where the inhibitor is seen in the complex.

Dieter Blaas and Michaela Schmidtke Research Groups

Significance

More than 160 rhinovirus (RV) types cause about a billion respiratory infections annually in the United States alone, contributing to influenza-like illness. This diversity makes vaccination impractical. Existing small-molecule inhibitors target RVs by binding to a hydrophobic pocket in the capsid but exhibit side effects, resistance, and/or mutational escape, impeding registration as drugs. The pyrazolopyrimidine OBR-5-340 acts like other capsid binders by preventing conformational changes required for genome release. However, by using cryo-EM, we show that OBR-5-340 inhibits the naturally pleconaril-resistant RV-B5 by attaching close to the pocket entrance in a binding geometry different from that of most capsid binders. Combinations of inhibitors with disparate binding modes might thus effectively combat RVs while reducing the risk of resistance development.

Wald, J.,  Pasin, M.,  Richter, M., Walther, C., Mathai, N., Kirchmair, J., Makarov, V. M., Goessweiner-Mohr, N., Marlovits, T. C., Zanella, I., Real-Hohn, A., Verdaguer, N., Blaas, D.,  and Schmidtke. M.: Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site, Proc. Natl. Acad. Sci. U.S.A. 2019, 116 (38), 19109-19115.https://doi.org/10.1073/pnas.1904732116

 

J. AM. CHEM. SOC. 2019

Nature Index Journal

 

Double-staining (PI/FAM) FCM analysis (A) and confocal microscopy images (B) of cells co-transfected with the (FAM)-MHDNA: netropsin (1:1) complex. In panel (A), the percentages of viable non-transfected cells, viable MH-DNA containing cells, dead/compromised non-transfected cells, and dead/compromised transfected cells are indicated in the bottom-left, bottom-right, top-left, and top-right quadrants, respectively. In panel (B), the green color marks the localization of (FAM)-MH-DNA, while the blue color marks cellc nuclei stained with Hoechst 33342. (C) Deconvoluted imino regions of 1D 1H NMR spectra of MH-DNA in vitro and the 1:1 MHDNA: netropsin complex in vitro and in cells. NMR spectra of extracellular fluid taken from the sample after in-cell NMR spectral acquisition and of non-transfected cells (cellular background) are shown in gray. The vertical green and blue dashed lines mark imino signals specific to the unbound and ligand-bound forms of MH-DNA, respectively.

Lukáš Trantírek Research Group

Significance

Studies on DNAligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNAligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNAligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cellNMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro -validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off -target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of druglike molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off -target binding.

Krafcikova, M., Dzatko, S., Caron, C., Granzhan, A., Fiala, R., Loja, T., Teulade-Fichou, M-P., Fessl, T., Hänsel-Hertsch, R., Mergny, J-L., Foldynova-Trantirkova, S., and Trantirek, L.: Monitoring DNA−Ligand Interactions in Living Human Cells Using NMR Spectroscopy, J. Am. Chem. Soc.  2019, 141, 13281-13825, DOI:10.1021/jacs.9b03031

 

More publications Research Highlights archive

Reader’s Corner

literature to read, science to follow

In this section, a distinct selection of six highly stimulating research publications and reviews published during past 6 months is presented. It is our hope that links to exciting science, which deserves attention of the structural biology community, will help you to locate gems in the steadily expanding jungle of scientific literature. You are welcome to point out to any paper you found interesting by sending a link or citation to readerscorner@ciisb.org. The section is being updated regularly.


 

Reader’s Corner Archive

Quote of October

“Siamo locomotive, non vagoni.”

Ivano Bertini (1940-2012)