Biology subjects

Barandun, J.

Publications and source records attributed to Barandun, J..

10 recordsLinked to original sources

bioRxiv2024

Nematocida displodere Mechanosensitive Ion Channel of Small Conductance 2 assembles into a unique 6-channel super-structure in vitro

Mechanosensitive ion channels play an essential role in reacting to environmental signals and sustaining cell integrity by facilitating ion flux across membranes. For obligate intracellular pathogens like microsporidia, adapting to changes in host environment is crucial for survival and propagation. Despite representing a eukaryote of extreme genome reduction, microsporidia have expanded the gene family of mechanosensitive ion channels of small conductance (mscS) through repeated gene duplication and horizontal gene transfer. All microsporidian genomes that are characterized to-date contain mscS genes of both eukaryotic and bacterial origin, and have at least 5 different mscS copies. Here, we investigated the cryo-electron microscopy structure of the bacterially derived mechanosensitive ion channel of small conductance 2 (MscS2) from Nematocida displodere, an intracellular pathogen of Caenorhabditis elegans. MscS2 is the most compact MscS known, and assembles into a unique superstructure in vitro with six heptameric MscS2 channels oligomerizing through their transmembrane domains. Individual MscS2 channels are oriented in a heterogeneous manner to one another, resembling an asymmetric, flexible six-way cross joint. Finally, we show that, despite the extreme compaction, microsporidian MscS2 still forms a heptameric membrane channel, conserving the most important structural features of bacterial MscS.

biochemistry↗

bioRxiv2023

Functional annotation of a divergent genome using sequence and structure-based homology.

BackgroundMicrosporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence homology. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes. ResultsIn this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin, allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted open reading frames, of which 89 % were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes. ConclusionWe provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

genomics↗

bioRxiv2023

Ribosome clustering and surface layer reorganization in the microsporidian host-invasion apparatus.

During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and demonstrates that ribosomes are efficiently transported through polar tubes in a spiral-like parallel arrangement.

biophysics↗

bioRxiv2022

Ribosomal protein with conserved function has entirely different structures in different organisms

Ribosomes from different species can markedly differ in their composition by including dozens of ribosomal proteins that are unique to specific lineages but absent in others. However, it remains unknown how ribosomes acquire and specialize new proteins throughout evolution. Here, to help answer this question, we describe the evolution of the ribosomal protein msL1/msL2 that was recently found in ribosomes from the parasitic microorganism clade, microsporidia. We first show that this protein has a conserved location in the ribosome but entirely dissimilar structures in different organisms: in each of the analyzed species, msL1/msL2 exhibits an altered secondary structure, an inverted orientation of the N- and C-termini on the ribosomal binding surface, and a completely transformed three-dimensional fold. We then show that this evolutionary fold switching is likely caused by changes in the msL1/msL2-binding site in the ribosome; specifically, by variations in microsporidian rRNA. These observations allow us to infer an evolutionary scenario in which a small, positively-charged, de novo-born unfolded protein was first captured by rRNA to become part of the ribosome and subsequently underwent complete fold switching to optimize its binding to its evolving ribosomal binding site. Overall, our work provides a striking example of how a protein can switch its fold in the context of a complex biological assembly while retaining its specificity for its molecular partner. This finding will help us better understand the origin and evolution of new protein components of complex molecular assemblies - thereby enhancing our ability to engineer biological molecules, identify protein homologs, and peer into the history of life on Earth.

evolutionary biology↗

bioRxiv2022

Reductive evolution in the structure of the microsporidian proteasome.

Proteasomes play an essential role in the life cycle of intracellular pathogens with extracellular stages by ensuring proteostasis in environments with limited resources. In microsporidia, divergent parasites with extraordinarily streamlined genomes, the proteasome complexity and structure are unknown, which limits our understanding of how these unique pathogens adapt and compact essential eukaryotic complexes. We present cryo-electron microscopy structures of the microsporidian 20S and 26S proteasome isolated from dormant or germinated Vairimorpha necatrix spores. The presence of distinct densities within the central cavity of the dormant spore proteasome suggests reduced activity in the environmental stage. In contrast, the absence of these densities and the existence of 26S particles post-germination indicates rapid reactivation of proteasomes after host infection. Structual and phylogenetic analyses reveal that microsporidian proteasomes have undergone extreme reductive evolution, lost three regulatory proteins, and compacted nearly every subunit. The highly derived microsporidian proteasome structure presented here reinforces the feasibility of the development of specific inhibitors and provides insight into the unique evolution and biology of these medically and economically important pathogens.

evolutionary biology↗

bioRxiv2021

Deep mutational scan of a drug efflux pump reveals its structure-function landscape

Drug efflux is a common resistance mechanism found in bacteria and cancer cells. Although several structures of drug efflux pumps are available, they provide only limited functional information on the phenomenon of drug efflux. Here, we performed deep mutational scanning (DMS) on the bacterial ATP binding cassette (ABC) transporter EfrCD to determine the drug efflux activity profile of more than 1500 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity towards positively charged ethidium, while additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryo-EM structure of EfrCD uncovered a high affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert the ABC exporter EfrCD into a drug-specific ABC importer.

biochemistry↗

bioRxiv2021

Protein-lipid interaction at low pH induces oligomerisation of the MakA cytotoxin from Vibrio cholerae

Many pathogenic bacteria produce protein toxins that target and perturb host cell membranes. The secreted -pore-forming toxins (-PFTs) cause membrane damage via pore formation. This study demonstrates a remarkable, hitherto unknown mechanism by an -PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other -PFTs. In contrast, MakA protein alone induces tube-like structures in the acidic lysosomal host cell compartment. In vitro studies unravel the dynamics of tubular growth, which occur in a pH-, lipid- and concentration-dependent manner. A 3.7-[A] cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. In its active -PFT conformation, MakA embeds its transmembrane helices into a thin annular lipid bilayer and spirals around a central cavity. Our study provides molecular insights into a novel tubulation mechanism of an -PFT protein, revealing a new mode of action by a secreted bacterial toxin.

cell biology↗

bioRxiv2020

Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome.

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-EM structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are universally highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which acts as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.

microbiology↗

bioRxiv2017

Modular assembly of the nucleolar large subunit processome

Early co-transcriptional events of eukaryotic ribosome assembly result in the formation of the small and large subunit processomes. We have determined cryo-EM reconstructions of the nucleolar large subunit processome in different conformational states at resolutions up to 3.4 [A]ngstroms. These structures reveal how steric hindrance and molecular mimicry are used to prevent premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Mutually exclusive conformations of these particles suggest that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly.

biochemistry↗

bioRxiv2017

The complete structure of the small subunit processome

The small subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small subunit processome at an overall resolution of 3.8 [A], which provides an essentially complete atomic model of this assembly. In this nucleolar superstructure, 51 ribosome assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry, steric hindrance as well as protein-and RNA-mediated RNA remodeling are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.

biochemistry↗