CIISB Research Highlights Archive

Interaction of Myxovalargin with the E. coli ribosome. (a) Relative position of P-site tRNA (green) and erythromycin (Ery, cyan) to 23S rRNA nucleotides protected from DMS by 10 μM (light blue) or 100 μM (dark blue) MyxB.

Andreas Kirschning, Daniel N. Wilson, and R. Müller Research Groups

Significance

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Koller, T.O., Scheid, U., et al.: The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition, J. Amer. Chem. Soc. (2023), 145, 851-863 https://doi.org/10.1021/jacs.2c08816

Features comparison between T4P-like structure obtained by cryo-EC, remote homology detection, and subtomogram averaging. The main dimensions of the T4P-like complex obtained by 3D cryo-EC (Center Left; EMD-14097), remote homology detection (Center), and subtomogram averaging (Center Right; EMD-14096) are compared. The Left and Right boxes show comparable images of the slice through at equivalent levels, from Top to Bottom, for the T4P-like complex obtained by cryo-EC (left box) and by cryo-ET subtomogram averaging (right box), both computed imposing a p6 symmetry. The dark-yellow boxes indicate the noncrystalline regions that are missing in the model obtained by cryo-EC with respect to the model obtained by subtomogram averaging. The S-layer (SL), the outer membrane (OM), and the inner membrane (IM) thicknesses are indicated. Scale bars indicate 50 Å.

Dario Piano Research Group

Significance

The cell envelope of the extremophile bacterium Deinococcus radiodurans was studied by cryo-electron microscopy and described with unprecedented detail. In this bacterium, the outermost cell envelope layer, named surface layer, is characterized by a highly regular tiling of proteins extending their crystalline organization to the cell envelope layers below (until the inner membrane). The study shows three main protein complexes, with masses in the MDa range, regularly organized into an astonishing geometrical regularity. The observed organization contributes to protecting the cell against environmental stressors and maintaining an efficient permeation of environmental solutes.

Surface layers (S-layers) are highly ordered coats of proteins localized on the cell surface of many bacterial species. In these structures, one or more proteins form elementary units that self-assemble into a crystalline monolayer tiling the entire cell surface. Here, the cell envelope of the radiation-resistant bacterium Deinococcus radiodurans was studied by cryo-electron microscopy, finding the crystalline regularity of the S-layer extended into the layers below (outer membrane, periplasm, and inner membrane). The cell envelope appears to be highly packed and resulting from a three-dimensional crystalline distribution of protein complexes organized in close continuity yet allowing a certain degree of free space. The presented results suggest how S-layers, at least in some species, are mesoscale assemblies behaving as structural and functional scaffolds essential for the entire cell envelope.

Farci, D., Haniewicz, P., and Piano, D.: The structured organization of Deinococcus radiodurans’ cell envelope, PNAS (2022) e112101, https://doi.org/10.1073/pnas.2209111119

Posttranslational modifications of tubulin are expected to control the functions of a wide range of microtubule-interacting proteins. Here, in vitro reconstitution assays with purified brain tubulin from mouse models lacking specific tubulin-modifying enzymes show that tubulin modification patterns selectively impact microtubule-protein interactions.

Carsten Janke and Marcus Braun Research Group

Significance
Tubulin posttranslational modifications have been predicted to control cytoskeletal functions by coordinating the molecular interactions between microtubules and their associating proteins. A prominent tubulin modification in neurons is polyglutamylation, the deregulation of which causes neurodegeneration. Yet, the underlying molecular mechanisms have remained elusive. Here, using in-vitro reconstitution, we determine how polyglutamylation generated by the two predominant neuronal polyglutamylases, TTLL1 and TTLL7, specifically modulates the activities of three major microtubule interactors: the microtubule-associated protein Tau, the microtubule-severing enzyme katanin and the molecular motor kinesin-1. We demonstrate that the unique modification patterns generated by TTLL1 and TTLL7 differentially impact those three effector proteins, thus allowing for their selective regulation. Given that our experiments were performed with brain tubulin from mouse models in which physiological levels and patterns of polyglutamylation were altered by the genetic knockout of the main modifying enzymes, our quantitative measurements provide direct mechanistic insight into how polyglutamylation could selectively control microtubule interactions in neurons.

Genova, M., Grycova, L., Puttrich, V., Magiera, M.M., Lansky, Z., Janke, C., and Braun, M.:

Tubulin polyglutamylation differentially regulates microtubule-interacting proteins

EMBO Journal, (2023) e112101, https://doi.org/10.15252/embj.2022112101

The mechanism of Renilla-type luciferase reaction and its inhibition.

Martin Marek and Zbyněk Prokop Research Group

Significance

The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago, but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type catalysis through crystallographic, spectroscopic and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing molecular snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O₂ via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase‒luciferin pairs for ultrasensitive optical bioassays.

Schenkmayerova, A., Toul, M., Pluskal, D., Baatallah, R., Gagnot, G., Pinto, G.P., Santana, V.T., Stuchla, M., Neugebauer, P., Chaiyen, P., Damborsky, J., Bednar, D., Janin, Y.L., Prokop, Z. & Marek, M.: Catalytic mechanism for Renilla-type luciferases, Nat. Catal. (2023). https://doi.org/10.1038/s41929-022-00895-z

A new technique for the digital mapping of biomarkers in tissues based on desorption and counting intact gold nanoparticle (Au NP) tags using infrared laser ablation single - particle inductively coupled plasma mass spectrometry (IR LA SP ICP MS).

Jan Preisler Research Group

Significance

In contrast to conventional UV laser ablation, Au NPs are not disintegrated during the desorption process due to their low absorption at 2940 nm. A mass spectrometer detects up to 83% of Au NPs. The technique is demonstrated on mapping a proliferation marker, nuclear protein Ki-67, in three-dimensional (3D) aggregates of colorectal carcinoma cells, and the results are compared with confocal fluorescence microscopy and UV LA ICP MS. Precise counting of 20 nm Au NPs with a single-particle detection limit in each pixel by the new approach generates sharp distribution maps of a specific biomarker in the tissue. Advantageously, the desorption of Au NPs from regions outside the tissue is strongly suppressed. The developed methodology promises multiplex mapping of low-abundant biomarkers in numerous biological and medical applications using multielemental mass spectrometers.

Stiborek, M., Jindřichová, L., Meliorisová, S., Bednařík, A., Prysiazhnyi, V., Kroupa, J., Houška, P., Adamová, B., Navrátilová, J., Kanický, V., and Preisler, J.:

Infrared Laser Desorption of Intact Nanoparticles for Digital Tissue Imaging

Anal. Chem. 2022 94, 51, 18114 – 18121, https://doi.org/10.1021/acs.analchem.2c05216

Labdane-type terpenes are a large family of bioactive natural products. Their often-complex structures present challenges to semisynthesis and de novo chemical synthesis in search of analogs with improved properties. To enable new modifications of the well-known tricyclic terpene forskolin, we have developed a distinct and potentially general synthetic scheme for the preparation of analogs of complex labdanes.

Jakub Švenda Research Group

Significance

We report a new synthetic strategy for the flexible preparation of forskolin-like molecules. The approach is different from the previously published works and employs a convergent assembly of the tricyclic labdane-type core from pre-functionalized cyclic building blocks. Stereoselective Michael addition enabled the fragment coupling with excellent control over three newly created contiguous stereocenters, all-carbon quaternary centers included. Silyl enol ether-promoted ring-opening metathesis paired with ring closure were the other key steps enabling concise assembly of the tricyclic core. Late-stage functionalization sequences transformed the tricyclic intermediates into a set of different forskolin-like molecules. The modular nature of the synthetic scheme described herein has the potential to become a general platform for the preparation of analogs of forskolin and other complex tricyclic labdanes.

Szczepanik, P. M., Mikhaylov, A. A., Hylse, O., Kučera, R., Daďová, P., Nečas, M., Kubala, L., Paruch, K., and Švenda, J.:

Convergent Assembly of the Tricyclic Labdane Core Enables Synthesis of Diverse Forskolin-like Molecules, Angew. Chem. Int. Ed. 2022, on-line version e202213183, https://doi.org/10.1002/anie.202213183