Biology subjects

Fislage, M.

Publications and source records attributed to Fislage, M..

7 recordsLinked to original sources

bioRxiv2026

From fibril to framework: P. abyssi AbpX illuminates a calcium-responsive family of microbial biomatrix proteins that form thermostable hydrogels

Evolutionary pressure on microbial communities propagating under extreme environmental conditions often results in unique structural adaptations to promote cell survival. Here, we report an investigation of AbpX, a biomatrix protein identified in cultures of the hyperthermophilic archaeon Pyrodictium abyssi. Under ex vivo and in vitro conditions, AbpX assembles into a para-crystalline lattice composed of semiflexible fibrils. CryoEM analysis of recombinant AbpX fibrils reveals that the precursor protein polymerizes through donor strand complementation (DSC), a process previously reported for chaperone-usher fimbriae in Gram-negative bacteria. Unlike the latter DSC protein polymers, AbpX undergoes chaperone-free polymerization in the presence of calcium ions, which are sequestered at the donor strand-acceptor groove interface between protomers in the fibril. Using a combination of cryoEM and crystallographic structural information, an atomic model is proposed for the AbpX lattice that provides insight into its potential role in biofilm formation. These findings suggest that calcium ion coordination triggers fibril assembly and pre-organizes the fibrils for incorporation into the protein lattice. Bioinformatic analysis indicates that AbpX exemplifies a distinct and broadly distributed clade of calcium ion responsive biomatrix proteins within the TasA superfamily that can be fabricated into hydrogel biomaterials in vitro under environmentally benign conditions. SignificanceBiofilms provide a protective environment for microbes that enhances resilience against environmental stressors. Secreted protein filaments constitute a major structural component of these extracellular matrices, however limited information is available on the mechanism of biofilm formation and structure of the resultant protein assemblies. Here, we report a class of biomatrix proteins that are widely distributed in bacteria and archaea. We demonstrate that one such protein, P. abyssi AbpX, self-assembles into fibrils and subsequently into para-crystalline lattices in response to the presence of calcium ion. We describe a mechanistic model for the structural evolution of the fibrils into an ordered protein framework that mimics the lattice structure of the ex vivo assembly observed during cell culture.

biophysics↗

bioRxiv2025

Cryo-EM analysis of the Bacillus thuringiensis extrasporal matrix identifies F-ENA as a widespread family of endospore appendages across the Firmicutes phylum

For over 100 years, Bacillus thuringiensis (Bt) has been used as an agricultural biopesticide to control pests caused by insect species in the orders of Lepidoptera, Diptera and Coleoptera. Under nutrient starvation, Bt cells differentiate into spores and associated toxin crystals that can adopt biofilm-like aggregates. We reveal that such Bt spore/toxin biofilms are embedded in a fibrous extrasporal matrix (ESM), and using cryoID, we resolved the structure and molecular identity of an uncharacterized type of pili, referred to here as Fibrillar ENdospore Appendages or F-ENA. F-ENA are monomolecular protein polymers tethered to the exosporium of Bt and are decorated with a flexible tip fibrillum. Phylogenetic analysis reveals that F-ENA is widespread not only in the class Bacilli, but also in the class Clostridia, and the cryoEM structures of F-ENA filaments from Bacillus, Anaerovorax and Paenibaccilus reveal subunits with a generic head-neck domain structure, where the {beta}-barrel neck of variable length latch onto a preceding head domain through short N-terminal hook peptides. In Bacillus, two collagen-like proteins (CLP) respectively tether F-ENA to the exosporium (F-Anchor), or constitute the tip fibrillum at the distal terminus of F-ENA (F-BclA). Sedimentation assays point towards F-ENA involvement in spore-spore clustering, likely mediated via F-BclA contacts and F-ENA bundling through the antiparallel interlocking of the head-neck units.

molecular biology↗

bioRxiv2024

Cryo-EM structures of the MnmE-MnmG complex reveal large conformational changes and provide new insights into the mechanism of tRNA modification

MnmE and MnmG form a conserved protein complex responsible for the addition of a 5-carboxymethylaminomethyl (cmnm5) group onto the wobble uridine of several tRNAs. Within this complex, both proteins collaborate intensively to catalyze a tRNA modification reaction that involves glycine as a substrate in addition to three different cofactors, with FAD and NADH binding to MnmG and methylenetetrahydrofolate (5,10-CH2-THF) to MnmE. Without structures of the MnmEG complex it remained enigmatic how these substrates and co-factors can be brought together in a concerted manner. Prior SAXS data suggested that the MnmE (2) and MnmG ({beta}2) homodimers can adopt either an 2{beta}2 or 4{beta}2 complex, depending on the nucleotide state of MnmE. Here, we report the cryo-EM structures of the MnmEG complex in the 2{beta}2 and 4{beta}2 oligomeric states. These structures reveal that MnmE undergoes large conformational changes upon interaction with MnmG, resulting in an asymmetric MnmE dimer. In particular, the functionally important C-terminal helix of MnmE relocates from the 5,10-CH2-THF binding pocket of MnmE to the FAD binding pocket of MnmG, thus suggesting a mechanism for the transfer of an activated methylene group from one active site to the other. Together, these findings provide crucial new insights into the MnmEG-catalyzed reaction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/625835v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@144e41forg.highwire.dtl.DTLVardef@1dce537org.highwire.dtl.DTLVardef@14c9359org.highwire.dtl.DTLVardef@5dec04_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

The structural scaffold of the TPLATE complex deforms the membrane during plant endocytosis

Summary paragraphEukaryotic cells maintain homeostasis of their outer membrane by controlled internalization of lipid and protein constituents via endocytosis1. Endocytosis is evolutionary conserved and utilizes similar structural folds. How these structural folds are combined into proteins and protein complexes however differs between eukaryotic kingdoms2. The TPLATE complex in plants is an evolutionary ancient protein module that combines several endocytic folds into a single octameric protein complex3-5. Its molecular architecture, lipid-nucleated condensate formation, and its requirement for clathrin cage curvature revealed its function in endocytosis initiation in plants6-8. Mechanistic understanding of how this complex drives membrane deformation during plant endocytosis is, however, lacking. Here, we used an integrative structural approach to obtain a precise molecular structure of the TPLATE complex. In addition, our approach allowed visualizing the structural flexibility that hallmarks this enigmatic complex. We prove that the intrinsic structural flexibility is required for its functionality and membrane recruitment. The membrane binding interface consists of several domains with differential lipid preferences. Finally, we show that the crescent shape of the structured part of the complex is sufficient for membrane curvature generation. Our mechanistic insight answers the long-standing question of how plants execute endocytosis without cytoskeletal-based force generation.

plant biology↗

bioRxiv2023

Structural insights in the GTP-driven monomerization and activation of a bacterial LRRK2 homologue using allosteric nanobodies

The Roco proteins are a family of GTPases, characterized by the conserved presence of a Roc-COR tandem domain. These proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinsons disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Recently, several structures of LRRK2 have been reported, but so far structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single particle cryo-EM to solve the structure of a simpler bacterial Roco protein (CtRoco) in its GTP-bound state, aided by the use of two conformation-specific nanobodies: NbRoco1 and NbRoco2. This structure presents CtRoco in an active monomeric state, featuring very significant conformational changes compared to the previously solved nucleotide-free dimer structure. In particular, the structure shows a very large GTP-induced conformational change of the LRR domain, unleashing it from the Roc-COR domains, using the LRR-Roc linker as a hinge. Furthermore, this structure shows how NbRoco1 and NbRoco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights in the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.

biochemistry↗

bioRxiv2023

A novel class of ultra-stable endospore appendages decorated with collagen-like tip fibrillae

Bacterial endospores are remarkable examples of biological resilience, representing a dormant and heavily fortified differentiation form capable of withstanding physical and chemical stressors detrimental to vegetative cells. In pathogenic firmicutes, spores also form an infectious particle and can take up a central role in the environmental persistence and dissemination of disease. A poorly understood aspect of spore-mediated infection is the fibrous structures or endospore appendages (ENAs) that have been seen to decorate the spores of pathogenic Bacilli and Clostridia. New methodological approaches are opening an unprecedented window on these long enigmatic structures. Using cryoID, Alphafold modelling and genetic approaches we identify a novel class of ultra-robust ENAs formed by Bacillus paranthracis. We demonstrate that L-ENA are encoded by a three-gene cluster (ena3) that contains all components for the self-assembly of ladder-like protein nanofibers of stacked heptameric rings, their anchoring to the exosporium, and their termination in a trimeric ruffle made of a collagen-like BclA paralogue. Phylogenomic analyses shows the ena3 gene cluster as a mobile element with a polyphyletic distribution across pathogenic Bacilli.

microbiology↗

bioRxiv2022

Blind spots on western blots: a meta-research study highlighting opportunities to improve figures and methods reporting

Western blotting is a standard laboratory method used to detect proteins and assess their expression levels. Unfortunately, poor western blot image display practices and a lack of detailed methods reporting can limit a readers ability to evaluate or reproduce western blot results. While several groups have studied the prevalence of image manipulation or provided recommendations for improving western blotting, data on the prevalence of common publication practices are scarce. We systematically examined 551 articles published in the top 25% of journals in neurosciences (n=151) and cell biology (n=400) that contained western blot images, focusing on practices that may omit important information. Our data show that most published western blots are cropped and blot source data are not made available to readers in the supplement. Publishing blots with visible molecular weight markers is rare, and many blots additionally lack molecular weight labels. Western blot methods sections often lack information on the amount of protein loaded on the gel, blocking steps and antibody labeling protocol. Important antibody identifiers like source, catalog number or RRID were omitted frequently for primary antibodies, and regularly for secondary antibodies. We present detailed descriptions and visual examples to help scientists, peer reviewers and editors to publish more informative western blot figures and methods. Additional resources include a toolbox to help scientists produce more reproducible western blot data, teaching slides in English and Spanish and an antibody reporting template.

cell biology↗