Overview of the dormant ribosome structure from 1 hpf zebrafish (a) and Xenopus egg (b). Ribosome-associated factors are shown as surface representations; eEF2b and eIF5a correspond to Protein Data Bank (PDB) accessions 6MTE and 5DAT, respectively.

Andrea Pauli Research Group

Significance

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4–eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

Leesch, F. et. al.: A molecular network of conserved factors keeps ribosomes dormant in the egg, Nature (2023), 613, 712-720: https://doi.org/10.1038/s41586-022-05623-y

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