CIISB Research Highlights Archive

Pavlina Rezacova Research Group

DNA binding to CggR in the cryo-EM structure. Model of the CggR–OLR biological unit fitted to a cryo-EM map.

Significance

The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.

Soltysova M. et al. Structural characterization of two prototypical repressors of SorC family reveals tetrameric assemblies on DNA and mechanism of function

Nucleic Acid Research 2024, 52(12), 7305-20, DOI: 10.1093/nar/gkae434

Radek Marek and Ondrej Jurcek Research groups

Structure-transformation analysis: reversible conversion reaction between Pd3(L1)6 and trimeric Pd3(L1)3Cl6 species suggesting the existence of a crown-like architecture.

Significance

Nature uses various chiral and unsymmetric building blocks to form substantial and complex supramolecular assemblies. In contrast, the majority of organic ligands used in metallosupramolecular chemistry are symmetric and achiral. Here we extend the group of unsymmetric chiral bile acids used as a scaffold for organic bispyridyl ligands by employing chenodeoxycholic acid (CDCA), an epimer of the previously used ursodeoxycholic acid (UDCA). The epimerism, flexibility, and bulkiness of the ligands leads to large structural differences in coordination products upon reaction with Pd(NO₃)₂. The UDCA-bispyridyl ligand self-assembles quantitatively into a single crown-like Pd₃L₆ complex, whereas the CDCA ligand provides a mixture of coordination complexes of general formula Pd_nL_2n, i.e., Pd₂L₄, Pd₃L₆, Pd₄L₈, Pd₅L₁₀, and even Pd₆L₁₂ containing an impressive 120 chiral centers. The coordination products were studied by a combination of analytical methods, with ion-mobility mass spectrometry (IM-MS) providing valuable details on their structure and allowed an effective separation of m/z 1461 to individual signals according to the arrival time distribution, thereby revealing four different ions of [Pd₃L₆(NO₃)₃]³⁺, [Pd₄L₈(NO₃)₄]⁴⁺, [Pd₅L₁₀(NO₃)₅]⁵⁺, and [Pd₆L₁₂(NO₃)₆]⁶⁺. The structures of all the complexes were modelled using DFT calculations. Finally, the challenges and conclusions in determining the specific structural identity of these unsymmetric species are discussed.

Jurcek O. et al. Unsymmetric Chiral Ligands for Large Metallo-Macrocycles: Selectivity of Orientational Self-Sorting

Angewandte Chemie International Edition 2024, 63(36), e202409134, DOI: 10.1002/anie.202409134

Pavel Plevka Research Group

Cryo-EM micrographs of the JBD30 virions binding to Type IV pili.

Significance

Bacteriophages are the most abundant biological entities on Earth, but our understanding of many aspects of their lifecycles is still incomplete. Here, we have structurally analysed the infection cycle of the siphophage Casadabanvirus JBD30. Using its baseplate, JBD30 attaches to Pseudomonas aeruginosavia the bacterial type IV pilus, whose subsequent retraction brings the phage to the bacterial cell surface. Cryo-electron microscopy structures of the baseplate-pilus complex show that the tripod of baseplate receptor-binding proteins attaches to the outer bacterial membrane. The tripod and baseplate then open to release three copies of the tape-measure protein, an event that is followed by DNA ejection. JBD30 major capsid proteins assemble into procapsids, which expand by 7% in diameter upon filling with phage dsDNA. The DNA-filled heads are finally joined with 180-nm-long tails, which bend easily because flexible loops mediate contacts between the successive discs of major tail proteins. It is likely that the structural features and replication mechanisms described here are conserved among siphophages that utilize the type IV pili for initial cell attachment.

Valentova L. et al. Structure and replication of Pseudomonas aeruginosa phage JBD30

EMBO Journal 2024, DOI: 10.1038/s44318-024-00195-1

A tetrastranded DNA structure, KNa-quadruplex (KNaQ), is described. KNaQ forms from repetitive DNA sequences that are abundant in (parasitic) worms but extremely rare in humans or livestock. This opens a possibility of exploiting the fold as a plausible antiparasitic drug target. The structure's unique properties distinguish it from all other known DNA quadruplexes and can be used to design novel recognition DNA elements/sensors.

Lukáš Trantírek and Martina Zivkovič Research Groups

Significance

DNA quadruplex structures provide an additional layer of regulatory control in genome maintenance and gene expression and are widely used in nanotechnology. We report the discovery of an unprecedented tetrastranded structure formed from a native G-rich DNA sequence originating from the telomeric region of Caenorhabditis elegans. The structure is defined by multiple properties that distinguish it from all other known DNA quadruplexes. Most notably, the formation of a stable so-called KNa-quadruplex (KNaQ) requires concurrent coordination of K+ and Na+ ions at two distinct binding sites. This structure provides novel insight into G-rich DNA folding under ionic conditions relevant to eukaryotic cell physiology and the structural evolution of telomeric DNA. It highlights the differences between the structural organization of human and nematode telomeric DNA, which should be considered when using C. elegans as a model in telomere biology, particularly in drug screening applications. Additionally, the absence/presence of KNaQ motifs in the host/parasite introduces an intriguing possibility of exploiting the KNaQ fold as a plausible antiparasitic drug target. The structure's unique shape and ion dependency and the possibility of controlling its folding by using low-molecular-weight ligands can be used for the design or discovery of novel recognition DNA elements and sensors.

Gajarsky, M. et al. DNA Quadruplex Structure with a Unique Cation Dependency,

Angew. Chem. Int. Ed. 2024, e202318261. https://doi.org/10.1002/anie.202313226

Slice through a tomogram of a stage-III ΔspoIVB B. subtilis sporangium, used for the segmentation (ii) of various forespore and mother cell ultrastructures. Panel ii shows the corresponding segmentation.

Christine Moriscot and Cecile Morlot Research Groups

Significance

Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilissporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins.

Bauda, E., Gallet, B., Moravcova, J. et al. Ultrastructure of macromolecular assemblies contributing to bacterial spore resistance revealed by in situ cryo-electron tomography.

Nat Commun 15, 1376 (2024). https://doi.org/10.1038/s41467-024-45770-6

Model for ameloblast-specific autoantibody production in patients with coeliac disease.

Jakub Abramson Research Group

Significance

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation—amelogenesis. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta. Defects in enamel formation are also found in patients with autoimmune polyglandular syndroze type-1 (APS-1), caused by AIRE deficiency, and in patients diagnosed with coeliac disease. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Gruper, Y., Wolff, A.S.B., Glanz, L. et al. Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Nature 624, 653–662 (2023). https://doi.org/10.1038/s41586-023-06776-0