CIISB Research Highlights Archive

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

Close-up view of FMA-binding pocket with residues creating the active site in stick representation. Key hydrogen bonds are shown as dashed yellow lines.

Martin Marek Research Group

Significance

NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O₂ via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

Nemergut, M., Pluskal, D., Horackova, J. et al. Illuminating the mechanism and allosteric behavior of NanoLuc luciferase.

Nat Commun 14, 7864 (2023). https://doi.org/10.1038/s41467-023-43403-y

Ultrahigh binding affinity of bambusuril receptors toward halides is achieved and continuously increases with increasing electron-withdrawing power of groups installed on its benzyl substituents, as measured by ¹H and ¹⁹F NMR spectroscopy.

Vladimír Šindelář Research Group

Significance

Inspired by nature, artificial hydrogen bond-based anion receptors have been developed to achieve high anion selectivity; however, their binding affinity is usually low. The potency of these receptors is usually increased by the introduction of aryl substituents, which withdraw electrons from their binding site through the resonance effect. Here, we show that the polarization of the C(sp³)-H binding site of bambusuril receptors, and thus their potency to bind anions, can be modulated by the inductive effect. The presence of electron-withdrawing groups on benzyl substituents of bambusurils significantly increases their binding affinities to halides, resulting in the strongest iodide receptor reported to date with an association constant greater than 10¹³ M⁻¹ in acetonitrile. A Hammett plot showed that while the bambusuril affinity toward halides linearly increases with the electron-withdrawing power of their substituents, their binding selectivity remains essentially unchanged.

Tuning CH Hydrogen Bond-Based Receptors toward Picomolar Anion Affinity via the Inductive Effect of Distant Substituents, M. Chvojka, D. Madea, H. Valkenier, V. Šindelář

Angew. Chem. Int. Ed. 2023, e202318261. https://doi.org/10.1002/anie.202318261