CIISB Research Highlights Archive | Czech Infrastructure for Integrative Structural Biology

Nature Communications 2025

RECQ4 cooperates with MUS81 to prevent ultrafine bridges formation.

Significance

Replication stress, particularly in hard-to-replicate regions such as telomeres and centromeres, leads to the accumulation of replication intermediates that must be processed to ensure proper chromosome segregation. In this study, we identify a critical role for the interaction between RECQ4 and MUS81 in managing such stress. We show that RECQ4 physically interacts with MUS81, targeting it to specific DNA substrates and enhancing its endonuclease activity. Loss of this interaction, results in significant chromosomal segregation defects, including the accumulation of micronuclei, anaphase bridges, and ultrafine bridges (UFBs). Our data further demonstrate that the RECQ4-MUS81 interaction plays an important role in ALT-positive cells, where MUS81 foci primarily colocalise with telomeres, highlighting its role in telomere maintenance. We also observe that a mutation associated with Rothmund-Thomson syndrome, which produces a truncated RECQ4 unable to interact with MUS81, recapitulates these chromosome instability phenotypes. This underscores the importance of RECQ4-MUS81 in safeguarding genome integrity and suggests potential implications for human disease. Our findings demonstrate the RECQ4-MUS81 interaction as a key mechanism in alleviating replication stress at hard-to-replicate regions and highlight its relevance in pathological conditions such as RTS.

Ashraf, et al.: RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

Nature Communications 2025, 10.1038/s41467-025-56518-1

JACS Au 2025

Loschmidt Laboratories

The scheme of the variational autoencoder-based pipeline for the design of novel sequences.

Significance

Enzymes play a crucial role in sustainable industrial applications, with their optimization posing a formidable challenge due to the intricate interplay among residues. Computational methodologies predominantly rely on evolutionary insights of homologous sequences. However, deciphering the evolutionary variability and complex dependencies among residues presents substantial hurdles. Here, we present a new machine-learning method based on variational autoencoders and evolutionary sampling strategy to address those limitations. We customized our method to generate novel sequences of model enzymes, haloalkane dehalogenases. Three design–build–test cycles improved the solubility of variants from 11% to 75%. Thorough experimental validation including the microfluidic device MicroPEX resulted in 20 multiple-point variants. Nine of them, sharing as little as 67% sequence similarity with the template, showed a melting temperature increase of up to 9 °C and an average improvement of 3 °C. The most stable variant demonstrated a 3.5-fold increase in activity compared to the template. High-quality experimental data collected with 20 variants represent a valuable data set for the critical validation of novel protein design approaches. Python scripts, jupyter notebooks, and data sets are available on GitHub (https://github.com/loschmidt/vae-dehalogenases), and interactive calculations will be possible via https://loschmidt.chemi.muni.cz/fireprotasr/.

Kohout, et al.: Engineering Dehalogenase Enzymes Using Variational Autoencoder-Generated Latent Spaces and Microfluidics

JACS Au 2025, 5(2), 10.1021/jacsau.4c01101

The FEBS Journal 2025

Lukáš Žídek Research Group

Electrostatic potential of unphosphorylated and phosphorylated MAP2c.

Significance

Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ.

Jansen, et al.: Characterization of multiple binding sites on microtubule associated protein 2c recognized by dimeric and monomeric 14‐3‐3ζ

The FEBS Journal, DOI: 10.1111/febs.17405

Nucleic Acid Research 2025

Gabriel Demo Research Group

Cryo-EM structure of 30S–30S dimer formed by aRDF protein.

Significance

Protein synthesis (translation) consumes a substantial proportion of cellular resources, prompting specialized mechanisms to reduce translation under adverse conditions. Ribosome inactivation often involves ribosome-interacting proteins. In both bacteria and eukaryotes, various ribosome-interacting proteins facilitate ribosome dimerization or hibernation, and/or prevent ribosomal subunits from associating, enabling the organisms to adapt to stress. Despite extensive studies on bacteria and eukaryotes, understanding factor-mediated ribosome dimerization or anti-association in archaea remains elusive. Here, we present cryo-electron microscopy structures of an archaeal 30S dimer complexed with an archaeal ribosome dimerization factor (designated aRDF), from Pyrococcus furiosus, resolved at a resolution of 3.2 Å. The complex features two 30S subunits stabilized by aRDF homodimers in a unique head-to-body architecture, which differs from the disome architecture observed during hibernation in bacteria and eukaryotes. aRDF interacts directly with eS32 ribosomal protein, which is essential for subunit association. The binding mode of aRDF elucidates its anti-association properties, which prevent the assembly of archaeal 70S ribosomes.

Hassan, et al.: Novel archaeal ribosome dimerization factor facilitating unique 30S–30S dimerization

Nucleic Acids Research 2025, 53(2), DOI: 10.1093/nar/gkae1324

Nature Communications 2024-2

Tomáš Kouba Research Group

Structural details of the highlighted MsmIMPDH monomer.

Significance

Allosteric regulation of inosine 5′-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential.

Bulvas, O., Knejzlík, Z., Sýs, J. et al. Deciphering the allosteric regulation of mycobacterial inosine-5′-monophosphate dehydrogenase.

Nat Commun 15, 6673 (2024). https://doi.org/10.1038/s41467-024-50933-6

Proceedings of the National Academy of Sciences of the United States of America 2024

GAG-binding sites of Sema2b. Ribbon representation of Sema2b^ΔC in complex with SOS, the sema domain is shown in orange, the PSI domain in blue, the Ig-like domain in red, and SOS is shown in stick representation. A 66-residue long C-terminal tail, which was omitted from the construct used for crystallization, is represented by a dashed gray line. The C-terminal central helix predicted by AlphaFold is shown in green. Schematic domain organization of Sema2b is shown below the ribbon representation, highlighting the locations of SOS binding sites with yellow stars. The high-affinity binding site is indicated by a violet star.

Daniel Rozbesky Research Group

Significance

The development of the nervous system relies on precise axon guidance, orchestrated by cues like semaphorins. These cues not only engage their cognate receptors but also interact with extracellular matrix components like proteoglycans, whose role remains poorly understood. Our study reveals that secreted semaphorins bind specifically to proteoglycan glycosaminoglycan chains via multiple sites, with an essential high-affinity site located at the C-terminal tail. Deleting this site disrupts semaphorin activity in guiding olfactory receptor neuron axons in vivo. We propose that secreted semaphorins attach to cell surfaces via proteoglycan interactions, aiding their recognition by axon receptors.

NOURISANAMI, Farahdokht; SOBOL, Margarita; LI, Zhuoran; HORVATH, Matej; KOWALSKA, Karolina et al. Molecular mechanisms of proteoglycan-mediated semaphorin signaling in axon guidance.

Proceedings of the National Academy of Sciences of the United States of America, 2024, e24202755121, https://doi.org/10.1073/pnas.2402755121