Bacteriophage-encoded chaperonins stimulate prion protein fibrillation in an ATP-dependent manner.

The pathogenesis of the various prion diseases is based on the conformational conversion of the prion protein from its physiological cellular form to the insoluble scrapie isoform. Several chaperones, including the Hsp60 family of group I chaperonins, are known to contribute to this transformation, but data on their effects are scarce and conflicting. In this work, two GroEL-like phage chaperonins, the single-ring OBP and the double-ring EL, were found to stimulate monomeric prion protein fibrillation in an ATP-dependent manner. The resulting fibrils were characterised by thioflavin T fluorescence, electron microscopy, proteinase K digestion assay and other methods. In the presence of ATP, chaperonins were found to promote the conversion of prion protein monomers into short amyloid fibrils with their further aggregation into less toxic large clusters. Fibrils generated with the assistance of phage chaperonins differ in morphology and properties from those formed spontaneously from monomeric prion in the presence of denaturants at acidic pH.

Structural Diversity of Tick-Borne Encephalitis Virus Particles in the Inactivated Vaccine Based on Strain Sofjin.

AbstractThe main approach to preventing tick-borne encephalitis (TBE) is vaccination. Formaldehyde-inactivated TBE vaccines have a proven record of safety and efficiency but have never been characterized structurally with atomic resolution. We report a cryoelectron microscopy (cryo-EM) structure of the formaldehyde-inactivated TBE virus (TBEV) of Sofjin-Chumakov strain representing the Far Eastern subtype. A 3.8  Šresolution reconstruction reveals the structural integrity of the envelope E proteins, specifically the E protein ectodomains. The comparative study shows high structural similarity to the previously published structures of the TBEV European subtype strains Hypr and Kuutsalo-14. A fraction of inactivated virions exhibits asymmetric features including the deformations of the membrane profile. We propose that the heterogeneity is caused by inactivation and perform a local variability analysis on the small parts of the envelope protein shell to reveal membrane curvature features possibly induced by the inactivation. The results of this study will have implications for design of novel vaccines against diseases caused by flaviviruses.

Nearly complete structure of bacteriophage DT57C reveals architecture of head-to-tail interface and lateral tail fibers.

The T5 family of viruses are tailed bacteriophages characterized by a long non-contractile tail. The bacteriophage DT57C is closely related to the paradigmal T5 phage, though it recognizes a different receptor (BtuB) and features highly divergent lateral tail fibers (LTF). Considerable portions of T5-like phages remain structurally uncharacterized. Here, we present the structure of DT57C determined by cryo-EM, and an atomic model of the virus, which was further explored using all-atom molecular dynamics simulations. The structure revealed a unique way of LTF attachment assisted by a dodecameric collar protein LtfC, and an unusual composition of the phage neck constructed of three protein rings. The tape measure protein (TMP) is organized within the tail tube in a three-stranded parallel α-helical coiled coil which makes direct contact with the genomic DNA. The presence of the C-terminal fragment of the TMP that remains within the tail tip suggests that the tail tip complex returns to its original state after DNA ejection. Our results provide a complete atomic structure of a T5-like phage, provide insights into the process of DNA ejection as well as a structural basis for the design of engineered phages and future mechanistic studies.

The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine ß-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain.

Regulatory adenine nucleotide-binding cystathionine ß-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect.

Fine Structure of Plasmodesmata-Associated Membrane Bodies Formed by Viral Movement Protein.

Cell-to-cell transport of plant viruses through plasmodesmata (PD) requires viral movement proteins (MPs) often associated with cell membranes. The genome of the Hibiscus green spot virus encodes two MPs, BMB1 and BMB2, which enable virus cell-to-cell transport. BMB2 is known to localize to PD-associated membrane bodies (PAMBs), which are derived from the endoplasmic reticulum (ER) structures, and to direct BMB1 to PAMBs. This paper reports the fine structure of PAMBs. Immunogold labeling confirms the previously observed localization of BMB1 and BMB2 to PAMBs. EM tomography data show that the ER-derived structures in PAMBs are mostly cisterns interconnected by numerous intermembrane contacts that likely stabilize PAMBs. These contacts predominantly involve the rims of the cisterns rather than their flat surfaces. Using FRET-FLIM (Förster resonance energy transfer between fluorophores detected by fluorescence-lifetime imaging microscopy) and chemical cross-linking, BMB2 is shown to self-interact and form high-molecular-weight complexes. As BMB2 has been shown to have an affinity for highly curved membranes at cisternal rims, the interaction of BMB2 molecules located at rims of adjacent cisterns is suggested to be involved in the formation of intermembrane contacts in PAMBs.

Can Activation of Acetylcholinesterase by ß-Amyloid Peptide Decrease the Effectiveness of Cholinesterase Inhibitors?

A central event in the pathogenesis of Alzheimer's disease (AD) is the accumulation of senile plaques composed of aggregated amyloid-ß (Aß) peptides. The main class of drugs currently used for the treatment of AD are the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. In this study, it has been shown that Aß augmented AChE activity in vitro, maximum activation of 548 ± 5% was achieved following 48 h of incubation with 10 µM of Aß1-40, leading to a 7.7-fold increase in catalytic efficiency. The observed non-competitive type of AChE activation by Aß1-40 was associated with increased Vmax and unchanged Km. Although BChE activity also increased following incubation with Aß1-40, this was less efficiently achieved as compared with AChE. Ex vivo electrophysiological experiments showed that 10 µM of Aß1-40 significantly decreased the effect of the AChE inhibitor huperzine A on the synaptic potential parameters.

DNA-Binding Protein Dps Protects Escherichia coli Cells against Multiple Stresses during Desiccation.

Gradual dehydration is one of the frequent lethal yet poorly understood stresses that bacterial cells constantly face in the environment when their micro ecotopes dry out, as well as in industrial processes. Bacteria successfully survive extreme desiccation through complex rearrangements at the structural, physiological, and molecular levels, in which proteins are involved. The DNA-binding protein Dps has previously been shown to protect bacterial cells from many adverse effects. In our work, using engineered genetic models of E. coli to produce bacterial cells with overproduction of Dps protein, the protective function of Dps protein under multiple desiccation stresses was demonstrated for the first time. It was shown that the titer of viable cells after rehydration in the experimental variants with Dps protein overexpression was 1.5-8.5 times higher. Scanning electron microscopy was used to show a change in cell morphology upon rehydration. It was also proved that immobilization in the extracellular matrix, which is greater when the Dps protein is overexpressed, helps the cells survive. Transmission electron microscopy revealed disruption of the crystal structure of DNA-Dps crystals in E. coli cells that underwent desiccation stress and subsequent watering. Coarse-grained molecular dynamics simulations showed the protective function of Dps in DNA-Dps co-crystals during desiccation. The data obtained are important for improving biotechnological processes in which bacterial cells undergo desiccation.

Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine.

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.

Silicon in sporoderms of micro- and megaspores of Isoetes echinospora Durieu registered by EDS and EELS.

The present study reveals silica in sporoderms of micro- and megaspores of the modern quillwort Isoetes echinospora Durieu and homologizes layers of the sporoderm in spores of this plant. Here, the presence of silica in sporoderms of microspores has been documented for the first time, and observations of megaspore sporoderms were used to test various methods, such as energy dispersive (EDS) and electron energy loss (EELS) spectroscopies. The results elucidate the occurrence of silicon in the quillworts and will influence on the interpretation of their modern and fossil sporoderms.

5-(Perylen-3-ylethynyl)uracil as an antiviral scaffold: Potent suppression of enveloped virus reproduction by 3-methyl derivatives in vitro.

Amphipathic nucleoside and non-nucleoside derivatives of pentacyclic aromatic hydrocarbon perylene are known as potent non-cytotoxic broad-spectrum antivirals. Here we report 3-methyl-5-(perylen-3-ylethynyl)-uracil-1-acetic acid and its amides, a new series of compounds based on a 5-(perylen-3-ylethynyl)-uracil scaffold. The compounds demonstrate pronounced in vitro activity against arthropod-borne viruses, namely tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV), in plaque reduction assays with EC50 values below 1.9 and 1.3 nM, respectively, and Chikungunya virus (CHIKV) in cytopathic effect inhibition test with EC50 values below 3.2 µM. The compounds are active against respiratory viruses as well: severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in cytopathic effect inhibition test and influenza A virus (IAV) in virus titer reduction experiments are inhibited - EC50 values below 51 nM and 2.2 µM, respectively. The activity stems from the presence of a hydrophobic perylene core, and all of the synthesized compounds exhibit comparable 1O2 generation rates. Nonetheless, activity can vary by orders of magnitude depending on the hydrophilic part of the molecule, suggesting a complex mode of action. A time-of-addition experiment and fluorescent imaging indicate that the compounds inhibit viral fusion in a dose-dependent manner. The localization of the compound in the lipid bilayers and visible damage to the viral envelope suggest the membrane as the primary target. Dramatic reduction of antiviral activity with limited irradiation or under treatment with antioxidants further cements the idea of photoinduced ROS-mediated viral envelope damage being the mode of antiviral action.

Mechanism of curaxin-dependent nucleosome unfolding by FACT.

Human FACT (FACT) is a multifunctional histone chaperone involved in transcription, replication and DNA repair. Curaxins are anticancer compounds that induce FACT-dependent nucleosome unfolding and trapping of FACT in the chromatin of cancer cells (c-trapping) through an unknown molecular mechanism. Here, we analyzed the effects of curaxin CBL0137 on nucleosome unfolding by FACT using spFRET and electron microscopy. By itself, FACT adopted multiple conformations, including a novel, compact, four-domain state in which the previously unresolved NTD of the SPT16 subunit of FACT was localized, apparently stabilizing a compact configuration. Multiple, primarily open conformations of FACT-nucleosome complexes were observed during curaxin-supported nucleosome unfolding. The obtained models of intermediates suggest "decision points" in the unfolding/folding pathway where FACT can either promote disassembly or assembly of nucleosomes, with the outcome possibly being influenced by additional factors. The data suggest novel mechanisms of nucleosome unfolding by FACT and c-trapping by curaxins.

Size Distribution of Inactivated Tick-Borne Encephalitis Virus Particles Revealed by a Comprehensive Physicochemical Approach.

Tick-borne encephalitis virus (TBEV) is an enveloped RNA virus, a member of the genus Flavivirus (family Flaviviridae). Here, we provide a detailed analysis of the size and structure of the inactivated TBEV vaccine strain Sofjin-Chumakov. Four analytical methods were used to analyze individual TBEV particles-negative staining TEM, cryo-EM, atomic force microscopy (AFM), and nanoparticle tracking analysis (NTA). All methods confirmed that the particles were monodisperse and that their mean size was ~50 nm. Cryo-EM data allowed us to obtain a 3D electron density model of the virus with clearly distinguishable E protein molecules. STEM-EELS analysis detected phosphorus in the particles, which was interpreted as an indicator of RNA presence. Altogether, the described analytical procedures can be valuable for the characterization of inactivated vaccine virus samples.

Application of Special Electron Microscopy Techniques to the Study of DNA - Protein Complexes in E. coli Cells.

Various electron microscopy techniques were applied recently to the study of DNA condensation in dormant bacterial cells. Here, we describe, in detail, the preparation of dormant Escherichia coli cells for electron microscopy studies and electron tomography and energy dispersive spectroscopy (EDS) approaches, which were used to reveal the structures of DNA-protein complexes in dormant Escherichia coli cells.

SERS-based biosensor with Raman-active external responsive element for rapid determination of adenosine monophosphate.

Phosphorylated adenosine derivatives are important biological molecules with diverse biological functions connected with the energetic balance of the cell, biosynthesis of cell components and regulation of protein activity. Measurement of these compounds provides information about the cell signalling in the body as well as the quantity of microorganisms in the environment. Surface-enhanced Raman spectroscopy (SERS) is an optical method that provides a unique spectrum of a substance at low concentrations. Specificity and limit of detection of SERS-based sensors can be increased drastically using nucleic acid aptamers and Raman-active dyes, respectively. Here we describe an adenosine monophosphate (AMP) biosensor based on AMP-dependent interaction between the well-known DNA aptamer for AMP and a novel Raman-active dye. The SERS intensity of novel Black Hole Quencher-2 (BHQ-2) derivatives was shown to be proportional to the charge of the molecule indicating electrostatic interactions with negatively charged colloidal silver nanoparticles. The novel derivative of BHQ-2 with two amine groups, BHQ-2-(NH2)2, binds an unpaired guanine stacked between guanine-guanine and guanine-adenine mismatches in DNA aptamer-AMP complex with KD = 26 nM as shown by 1H nuclear magnetic resonance, molecular docking and biolayer interferometry. The aptamer is pre-structured by AMP being folded in the conformation favorable for the interaction with BHQ-2-(NH2)2. This specific mechanism of the interaction allows designing of a SERS-based aptasensor with a limit of detection being as low as 3.4 nM of AMP and the dynamic range of nearly 5 orders - from 3.4 nM to 200 µM. The results illustrate a new approach to biosensors where DNA-interacting ligands act as external responsive elements providing an analyte-dependent SERS signal.

Effect of bacteriophage-encoded chaperonins on amyloid transformation of α-synuclein.

Controversial information about the role of chaperonins in the amyloid transformation of proteins and, in particular, α-synuclein, requires a more detailed study of the observed effects due to the structure and functional state of various chaperonins. In this work, two types of phage chaperonins, the double-ring EL and the single-ring OBP, were shown to stimulate α-synuclein fibrillation in an ATP-dependent manner. Chaperonin morphology does not affect the stimulation of α-synuclein amyloid transformation. However, the ATP-dependent effect of single- and double-ring chaperonins on this process differs, which can lead to different morphology of resulting fibrils. Fibril formation seems to proceed without substrate encapsulation in the internal cavity of chaperonin, because of the structural features of phage chaperonins and their ability to function without co-chaperonins. In the absence of ATP, both chaperonins, on the contrary, completely prevent α-synuclein amyloid transformation, which provides the possibility of their use as anti-amyloid agents, in the form of incomplete molecules or mutants with suppressed ATPase activity.

Structural characterization of ß-propiolactone inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles.

The severe COVID-19 pandemic drives the research toward the SARS-CoV-2 virion structure and the possible therapies against it. Here, we characterized the ß-propiolactone inactivated SARS-CoV-2 virions using transmission electron microscopy (TEM) and atomic force microscopy (AFM). We compared the SARS-CoV-2 samples purified by two consecutive chromatographic procedures (size exclusion chromatography [SEC], followed by ion-exchange chromatography [IEC]) with samples purified by ultracentrifugation. The samples prepared using SEC and IEC retained more spikes on the surface than the ones prepared using ultracentrifugation, as confirmed by TEM and AFM. TEM showed that the spike (S) proteins were in the pre-fusion conformation. Notably, the S proteins could be recognized by specific monoclonal antibodies. Analytical TEM showed that the inactivated virions retained nucleic acid. Altogether, we demonstrated that the inactivated SARS-CoV-2 virions retain the structural features of native viruses and provide a prospective vaccine candidate.

Conservative and Atypical Ferritins of Sponges.

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.

The Structure of the Potato Virus A Particles Elucidated by Small Angle X-Ray Scattering and Complementary Techniques.

Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVAΔ32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVAΔ32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55°C was also different. The data of transmission electron microscopy showed that the PVAΔ32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen.

Fiber-Like Organization as a Basic Principle for Euchromatin Higher-Order Structure.

A detailed understanding of the principles of the structural organization of genetic material is of great importance for elucidating the mechanisms of differential regulation of genes in development. Modern ideas about the spatial organization of the genome are based on a microscopic analysis of chromatin structure and molecular data on DNA-DNA contact analysis using Chromatin conformation capture (3C) technology, ranging from the "polymer melt" model to a hierarchical folding concept. Heterogeneity of chromatin structure depending on its functional state and cell cycle progression brings another layer of complexity to the interpretation of structural data and requires selective labeling of various transcriptional states under nondestructive conditions. Here, we use a modified approach for replication timing-based metabolic labeling of transcriptionally active chromatin for ultrastructural analysis. The method allows pre-embedding labeling of optimally structurally preserved chromatin, thus making it compatible with various 3D-TEM techniques including electron tomography. By using variable pulse duration, we demonstrate that euchromatic genomic regions adopt a fiber-like higher-order structure of about 200 nm in diameter (chromonema), thus providing support for a hierarchical folding model of chromatin organization as well as the idea of transcription and replication occurring on a highly structured chromatin template.