Daphna Fenel

Bacterial spores owe their remarkable resistance properties to a multilayered coat, one of the most resilient and durable biological structures on Earth. Assembled at the surface of the outer forespore membrane, the coat comprises dozens of proteins organized into distinct layers. Its formation is initiated by SpoIVA, which is proposed to form a polymeric scaffold for the innermost coat layer. Although SpoIVA has been shown to polymerize into filaments in vitro , there is currently no evidence demonstrating the formation of such assemblies in vivo , and the mechanism underlying its oligomerization remains unresolved. In this study, cryo-focused ion beam milling combined with cryo-electron tomography of sporulating Bacillus subtilis cells reveals that the SpoIVA layer consists of polymers that form track-like structures radiating from the mother cell-proximal forespore pole and extending directionally toward the distal pole. Subtomogram averaging further sheds light on their organized architecture, harboring a straight orientation, uniform spacing, and embedding in the outer forespore membrane. These observations also define SpoIVA spatial orientation relative to the outer forespore membrane. Furthermore, AlphaFold3 predictions, combined with biophysical and functional assays, show that SpoIVA dimerizes through its central and C-terminal regions. We further show that dimerization promotes SpoIVA localization around the forespore but is dispensable for polymer formation, which relies on the ATPase domain. Altogether, these findings suggest a dual oligomerization mechanism, in which SpoIVA transitions from dimers to linear track-like polymers, and reveal that these assemblies play critical roles in coat assembly and spore development.

M42 peptidases (TET) are ubiquitous, giant self-compartmentalized aminopeptidases forming unique tetrahedral structures. TETs may participate in protein homeostasis and amino acid recycling by processing peptides downstream of the proteasome and other related proteolytic complexes. Currently, only five TETs have been extensively studied in Archaea from the Thermococcales order, with a predominant focus on structural characterization. Therefore, a clear view of the diversity and biological activities of this large enzymatic family is lacking. In this study, we establish robust criteria for high-throughput identification of TET peptidases from sequence data and reveal their wide taxonomic distribution in Archaea. We functionally characterize six additional TETs, including four from Asgardarcheota. Our results identify two main functional classes, with generalist TETs displaying broad-spectrum activities, and specialized TETs with more selective activities. We also report the first description of methionyl- and isoleucyl-aminopeptidases in the TET family. In addition to their diverse substrate specificities, the characterized TETs exhibit distinct activation profiles, varying in optimal temperature, pH, and metal cofactor requirements. Combining functional and phylogenetic analyses, we propose a classification of TET peptidases into 11 groups. Notably, we identify generalist group 11 as the ancestral TET in Archaea, from which specialized TETs arose following gene duplication or horizontal transfer. Finally, we highlight the presence of multiple TETs with selective activities in heterotrophic and mixotrophic organisms, suggesting a metabolic role for these enzymes in the degradation of environmental peptides. Overall, our work illuminates an underexplored diversity of TET enzymes, uncovering a complex evolutionary history that shaped their functional diversification

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N -acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains with their catalytic sites facing in opposite directions. Along with in vitro activity assays using fluorescently labeled and chemically defined substrates, these findings provide a molecular insight into donor substrate recognition and demonstrate that the glycosyltransferase reactions are highly specific. A combination of in vitro and in cellulo mutational studies was used to dissect the functional role of the four catalytic sites. While EXT1 is able to catalyze both glycosyltransferase reactions, EXT2 harbors only N -acetylglucosamine transferase activity. Our results provide mechanistic insight into heparan sulfate chain elongation as a non processive process and lay the cornerstone for future studies on EXT1-EXT2 function in health and disease.

Chaperones, as modulators of protein conformational states, are key cellular actors to prevent the accumulation of fibrillar aggregates. Here, we integrated kinetic investigations with structural studies to elucidate how the ubiquitous co-chaperonin prefoldin (PFD) inhibits diabetes associated islet amyloid polypeptide (IAPP) fibril formation. We demonstrated that both human and archaeal PFD interfere similarly with the IAPP fibril elongation and secondary nucleation pathways. Using archaeal prefoldin model, we combined NMR spectroscopy with EM to establish that the inhibition of fibril formation is mediated by the binding of prefoldin’s coiled-coil helices to the flexible IAPP N-terminal segment accessible on the fibril surface and fibril ends. AFM demonstrates that binding of prefoldin to IAPP leads to the formation of lower amounts of aggregates, composed of shorter fibrils, clustered together. Linking structural models with observed fibrillation inhibition processes opens new perspectives for understanding the interference between natural chaperones and formation of disease-associated amyloids.