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DNA gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, a detailed architecture of the full-length DNA gyrase from the model organism E . coli is still missing. Herein, we report the complete structure of the E . coli DNA gyrase nucleoprotein complex trapped by the antibiotic gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of two states of the DNA-binding/cleavage domain provides a better understanding of the allosteric movements of the enzyme complex. The local atomic resolution in the DNA-bound area reaching up to 3.0 Å enables the identification of the antibiotic density. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates. Bacterial DNA gyrase is the only type II DNA topoisomerase capable of introducing negative supercoils into DNA and is of interest as a drug target. Here the authors present the cryo-EM structure of the complete E . coli DNA gyrase bound to a 180 bp double-stranded DNA and the antibiotic gepotidacin, which reveals the connections between the functional domains and their spatial organization.

Liquid–liquid phase separation of multivalent intrinsically disordered protein–RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA–protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA–protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA–protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein–RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein−RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein−RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

Enabled by long-read sequencing technologies, particularly Single Molecule, Real-Time sequencing, N 6 -methyladenine (6mA) footprinting is a transformative methodology for revealing the heterogenous and dynamic distribution of nucleosomes and other DNA-binding proteins. Here, we present ipdTrimming, a novel 6mA-calling pipeline that outperforms existing tools in both computational efficiency and accuracy. Utilizing this optimized experimental and computational framework, we are able to map nucleosome positioning and transcription factor occupancy in nuclear DNA and establish high-resolution, long-range binding events in mitochondrial DNA. Our study highlights the potential of 6mA footprinting to capture coordinated nucleoprotein binding and to unravel epigenetic heterogeneity.

The dynamic interplay between a multimeric phosphoprotein (P) and polymeric nucleoprotein (N) in complex with the viral RNA is at the heart of the functioning of the RNA-synthesizing machine of negative-sense RNA viruses of the order Mononegavirales. P multimerization and N phosphorylation are often cited as key factors in regulating these interactions, but a detailed understanding of the molecular mechanisms is not yet available. Working with recombinant rabies virus (RABV) N and P proteins and using mainly surface plasmon resonance, we measured the binding interactions of full-length P dimers and of two monomeric fragments of either circular or linear N-RNA complexes, and we analyzed the equilibrium binding isotherms using different models. We found that RABV P binds with nanomolar affinity to both circular and linear N-RNA complexes and that the dimerization of P protein enhances the binding affinity by 15–30-fold as compared to the monomeric fragments, but less than expected for a bivalent ligand, in which the binding domains are connected by a flexible linker. We also showed that the phosphorylation of N at Ser389 creates high-affinity sites on the polymeric N-RNA complex that enhance the binding affinity of P by a factor of about 360.

In this review, we summarize computational and experimental data gathered so far showing that structural disorder is abundant within paramyxoviral nucleoproteins (N) and phosphoproteins (P). In particular, we focus on measles, Nipah, and Hendra viruses and highlight both commonalities and differences with respect to the closely related Sendai virus. The molecular mechanisms that control the disorder-to-order transition undergone by the intrinsically disordered C-terminal domain (NTAIL) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins are described in detail. By having a significant residual disorder, NTAIL-XD complexes are illustrative examples of \"fuzziness\", whose possible functional significance is discussed. Finally, the relevance of N-P interactions as promising targets for innovative antiviral approaches is underscored, and the functional advantages of structural disorder for paramyxoviruses are pinpointed.

Lassa fever hemorrhagic virus (LFHV) outbreaks in Nigeria continue to increase. With the possibility of a potentially deadly pandemic and the absence of a vaccine, an effective candidate drug that presents with no adverse effects and better clinical outcomes is urgently needed; hence, the aim of the study. Retrieved ligands: 5-(Methylene) evodiamine, N -(2-Fluorobenzoyl) evodiamine, N -[[2-(3-chlorophenyl)-5-(trifluoromethyl) pyrazol-3-yl]methyl]-2-(1-methyl-2,3-dihydroindol-5-yl) propanamide (NCTPMMDP), lauric acid, and sofosbuvir, and the target protein, 3MX5, were prepared and utilised for docking, molecular dynamics simulation, ADMET profiling, and DFT using standard protocols. The docking score for lauric acid was the lowest, with a value of − 6.1, followed by the standard drug and co-crystal control drug with scores of − 9.1 and − 8.9 kcal/mol, respectively. On the other hand, the scores of the other ligands ranged from − 9.5 to − 12.3 kcal/mol. Molecular simulations revealed fluctuations in the RMSD and RMSF values; however, overall, the formed complexes were stable during most of the docking simulation runs. Principal component analysis (PCA) analysis showed that all the ligands, apart from lauric acid, showed better conformational changes within the backbone of the protein. ADMET profiling showed that the ligands were better oral drug candidates than sofosbuvir, as revealed by their better absorption values and absolute compliance with the Lipinski rule of five. The DFT result showed that, apart from lauric acid, all the test ligands possess small energy gaps indicative of high reactivity. Put together, these findings indicate that the test ligands possess anti-LHFV potential that merits further in vivo and in vitro evaluations.

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (N TAIL ) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of N TAIL that binds P is situated 90 amino acids from the folded RNA-binding domain (N CORE ) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of N TAIL in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that N TAIL is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with N CORE . We present a model in which the first 50 disordered amino acids of N TAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive N CORE helical turns. The model provides a structural framework for understanding the role of N TAIL in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.

Background The freshwater snails Biomphalaria alexandrina and Bulinus trancatus are key contributors to the transmission of S. mansoni and S. haematobium , respectively, for being their intermediate hosts. Objectives This research study aimed to investigate the potency of the nucleoproteins (NPs) extracted from both snail species on the host immune reactions as an approach to developing a potential vaccine. Methods Three groups of six-week-old Swiss-Webster mice ( n  = 18; 15–20 g each) were injected intraperitoneally for three consecutive weeks with single doses (once a week) of B. alexandrina , B. truncatus , or a mixture of their nucleoproteins (50 µg each). On day 21st, the nucleoprotein-treated mice altogether, with six more mice, received subcutaneously S. mansoni cercariae (60/mouse). Eight weeks later, the experimental mice were sacrificed for evaluation of certain parasitological, molecular and immunological responses. Results The data of mice immunized with the various types of nucleoproteins showed a significant increase of FAS/R gene expressions in hepatic tissues and anti-IgG antibody levels in sera on the one hand and a significant decrease of worm loads and β-actin/R gene expression levels on the other hand when compared to the infected control mice. Conclusion These findings highlight the role of snails in immunomodulation and shed light on the possibility of antagonizing effects that might occur when the nucleoproteins of different species are mixed. Moreover, this research study might promote the literature spotting the importance of snail proteins against schistosomiasis.

The Crimean Congo virus has been reported to be a part of the spherical RNA-enveloped viruses from the Bunyaviridae family. Crimean Congo fever (CCHF) is a fatal disease with having fatality rate of up to 40%. It is declared endemic by the World Health Organization. Many outbreaks of CCHF have been reported over the years. Former studies on CCHF have reported that the nucleoprotein of CCHF, being a pivotal protein in the replication process of the virus, is a potential target for antiviral drugs. However, there is no specific drug that can be used to treat this fatal disease and laboratory testing is prohibited due to its pathogen level 4. This study aims to find a possible potential inhibitor of the nucleoprotein of CCHFV using modern techniques leading ultimately to the development of effective and natural drugs. In this study, a virtual screening procedure involving a docking process followed by the Molecular Dynamics method is used to find out the potential inhibitors of the nucleoprotein of CCHFV. Phytochemicals having pharmacological properties and approved by the Food and Drug Administration are docked over the nucleoprotein of CCHFV. The study signifies the use of Withanolide E as a drug for the treatment of CCHFV as the study depicts the potential of Withanolide E to inhibit the nucleoprotein of CCHFV using reliable and modern techniques.