Andrey L. Konevega

This work presents DSSNA (Define Spatial Structure of Nucleic Acids) v2026, a free and open-source standalone GROMACS[1] module for the automated analysis of nucleic acid structures from atomic coordinates that reproduces the core functionality of X3DNA[2] and DSSR[3] while extending it to the analysis of molecular dynamics (MD) trajectories. The algorithm identifies and classifies canonical and non-canonical base pairs, hydrogen-bond networks, stacking interactions, helices, and other elements of nucleic acid secondary and tertiary structure, using neighbor-search algorithms for efficient analysis of large trajectory datasets and a modular output system that lets users select the structural metrics required for a particular analysis. Application to a 1 µ s MD trajectory of a GUAA tetraloop mutant of the sarcin–ricin domain of Escherichia coli 23S ribosomal RNA (PDB: 1MSY) showed that persistent canonical base pairs form the structural core that maintains the secondary-structure profile, whereas most non-canonical base pairs and stacking interactions are transient. These results demonstrate that DSSNA enables the characterization of both static nucleic acid structures and the temporal evolution of structural interactions in MD simulations. Abstract Figure:

RelA/SpoT homologue family enzymes participate in controlling the cellular levels of the alarmone (p)ppGpp, thereby activating the stringent response and promoting survival under stress conditions. These proteins contain an N-terminal catalytic domain and a C-terminal regulatory domain. They catalyze both the synthesis of ppGpp/pppGpp from ATP and GDP/GTP and their hydrolysis to GDP/GTP and pyrophosphate. Here, we report the crystal structure of the N-terminal domain of Rel from Streptococcus equisimilis in complex with pppGpp at 3.2 Å resolution. The asymmetric unit contains a dimer with asymmetric ligation: pppGpp occupies only the synthetase site in one monomer, whereas in the other monomer, it is bound in both the hydrolase and synthetase sites. The two monomers exhibit distinct conformational states, with pronounced rearrangements of the flexible loops surrounding the binding pockets, including the α2/α3 and α8/α9 loops that act as steric gates. Molecular dynamics simulations support the dual binding arrangement and reveal additional probable transient binding sites, including a region in the linker between hydrolase and synthetase subdomains. These findings provide a structural framework for understanding how pppGpp binding modulates the opposing catalytic activities of bifunctional Rel enzymes and suggest possible mechanisms for (p)ppGpp-mediated autoregulation.

In prokaryotes, translation initiation orchestrates protein synthesis through a network of dynamic interactions among the ribosome, mRNA, initiator tRNA^fMet, and initiation factors (IFs). Traditional approaches that rely on radioactive labeling or surface immobilization are hindered by inherent safety risks and methodological constraints. We present a fluorescence-based analytical platform that integrates microscale thermophoresis (MST) as a unified, multiparametric toolkit for comprehensive interrogation of bacterial translation initiation at the molecular level. By systematically applying MST to a panel of fluorescently labeled components—initiator tRNA^fMet, mRNAs, and initiation factors—we quantify assembly pathways and equilibria as initiation progresses from simple bimolecular interactions to higher-order, multicomponent complexes. To broaden the fluorescence toolbox for ribosomal studies, we developed a robust BODIPY-labeling protocol for 70S ribosomes and confirmed preservation of structural integrity and function by nano differential scanning fluorimetry, stopped-flow kinetic assays, and peptide-synthesis activity tests. Our microscale fluorescent system facilitates probing initiation at a variety of steps, since the role of magnesium ions and initiation factors upon 30S initiation complex formation. The same platform can be applied to investigate the effects of different compounds on translation initiation, as demonstrated for a number of antibiotics, aptamers, and antimicrobial peptides. Using this approach, we determined the antibiotic streptomycin dissociation constant for both 30S and 70S ribosomes, which proved identical at 0.3 ± 0.1 μM, and demonstrated the effect of the antimicrobial peptide rumicidin-1 on translation initiation. Offering a cost-effective and high-sensitivity alternative to conventional methods, this approach advances mechanistic understanding of prokaryotic translation and provides a versatile framework for the discovery of novel protein synthesis inhibitors.

Rel/SpoT family enzymes participate in controlling the cellular levels of the alarmone (p)ppGpp, thereby activating the stringent response and promoting survival under stress conditions. These proteins contain an N-terminal catalytic domain and a C-terminal regulatory domain. They catalyze both the synthesis of ppGpp/pppGpp from ATP and GDP/GTP and their hydrolysis to GDP/GTP and pyrophosphate. Here, we report the crystal structure of the N-terminal domain of Rel from Streptococcus equisimilis (Rel_Seq385) in complex with pppGpp at 3.2 Å resolution. The asymmetric unit contains a dimer with asymmetric ligation, in which pppGpp occupies only the synthetase site in one monomer, whereas it is observed in both the hydrolase and synthetase sites in the other. Molecular dynamics simulations supported this binding arrangement for the monomer with both sites occupied and revealed additional probable transient binding sites that may contribute to alarmone binding.

In prokaryotes translation initiation orchestrates protein synthesis through a network of dynamic interactions among the ribosome, mRNA, initiator tRNAfMet, and initiation factors (IFs). Traditional approaches that rely on radioactive labeling or surface immobilization are hindered by inherent safety risks and methodological constraints. We present a fluorescence-based analytical platform that integrates microscale thermophoresis (MST) to investigate translation initiation at the molecular level. Employing fluorescently labeled molecules including the initiator tRNAfMet, mRNA, and Ifs, enabled a detailed characterization of initiation complex assembly as it progresses from bimolecular to higher-order multicomponent states. To expand the fluorescent toolbox for translation studies we established a novel BODIPY-labeling protocol for 70S ribosomes and confirmed their conformational integrity using nano differential scanning fluorimetry (nanoDSF). Our microscale fluorescent system facilitates probing initiation at a variety of steps, since the role of magnesium ions and initiation factors upon 30S initiation complex formation. The same platform can be applied to investigate the effects of different compounds on translation initiation, as demonstrated for a number of antibiotics, aptamers, and antimicrobial peptides. Using this approach, we determined the antibiotic streptomycin dissociation constant for both 30S and 70S ribosomes, which proved identical at 0.3 mcM, and demonstrated the effect of the antimicrobial peptide rumicidin-1 on translation initiation. Offering a cost-effective and high-sensitivity alternative to conventional methods, this approach advances mechanistic understanding of prokaryotic translation and provides a versatile framework for the discovery of novel protein synthesis inhibitors.

The development of highly sensitive approaches for detecting tumor cells in biological samples remains a critical challenge in laboratory and clinical oncology. In this study, we investigated the structural and magnetic properties of iron oxide nanoparticles incorporated into cellulose microspheres of two size ranges (~100 and ~700 μm) and evaluated their potential for targeted tumor cell isolation. In the smaller microspheres, magnetite-based magnetic nanoparticles (MNPs) were synthesized in situ via co-precipitation, whereas pre-synthesized MNPs were embedded into the larger microspheres. The geometrical characteristics of the resulting magnetic cellulose microspheres (MSCMNs) were assessed by confocal microscopy. Transmission electron microscopy and X-ray diffraction analyses revealed an average magnetic core size of approximately 17 nm. Magnetic properties of the MNPs within MSCMNs were characterized using a highly sensitive nonlinear magnetic response technique, and their dynamic parameters were derived using a formalism based on the stochastic Hilbert–Landau–Lifshitz equation. To evaluate their applicability in cancer diagnostics and treatment monitoring, the MSCMNs were functionalized with a TKD peptide that selectively binds membrane-associated Hsp70 (mHsp70), yielding TKD@MSCMNs. Magnetic separation enabled the isolation of tumor cells from biological fluids. The specificity of TKD-mediated binding was confirmed using Flamma648-labeled Hsp70 and compared with control alloferone-conjugated microspheres (All@MSCMNs). The ability of TKD@MSCMNs to selectively extract mHsp70-positive tumor cells was validated using C6 glioma cells and mHsp70-negative FetMSCs controls. Following co-incubation, the extraction efficiency for C6 cells was 28 ± 14%, significantly higher than that for FetMSC (7 ± 7%, p < 0.05). These findings highlight the potential of TKD-functionalized magnetic cellulose microspheres as a sensitive platform for tumor cell detection and isolation.

Under adverse environmental conditions bacterial cells survive by activating the “stringent response,” which is mediated by the accumulation of small signaling alarmone molecules (p)ppGpp. The synthesis of (p)ppGpp from ATP and GDP/GTP and their hydrolysis to GDP/GTP and pyrophosphate are catalyzed by enzymes of the RSH (RelA/SpoT Homologue) family. Studying the complex allosteric mechanism underlying the bifunctional activity of these enzymes and the maintenance of intracellular levels of (p)ppGpp is a topical issue in modern science. In this work we determine the conformational stability and binding efficiency of the bifunctional homologue of the RSH family Rel_Seq385 with nucleotides depending on their concentration and the ionic composition of the reaction mixture.

The rise of antimicrobial resistance among pathogenic bacteria poses a critical challenge to modern medicine, highlighting an urgent need for novel therapeutic agents. Bottromycin A ₂ (BotA2) is a promising candidate for future drug development, demonstrating potent activity against clinically relevant pathogens, including methicillin-resistant Staphylococcus aureus , vancomycin-resistant Enterococcus , and Mycoplasma species, although its molecular mechanism of action has remained unclear until now. Here, we demonstrate that BotA2 inhibits bacterial translation with unique context specificity determined by the mRNA coding sequence. Using high-throughput toe-printing coupled with deep sequencing (Toe-seq analysis), we show that BotA2 induces ribosome pausing predominantly when a glycine codon enters the A-site of the ribosome, regardless of the codon identities in the P- and E-sites. Our biochemical and biophysical data indicate that BotA2 specifically arrests glycine-delivering ternary complexes on the ribosome, thereby preventing full accommodation of incoming Gly-tRNA ^Gly within the peptidyl transferase center. Altogether, our findings uncover a previously undescribed mechanism of translation inhibition, driven by the context-specific immobilization of ternary complexes on elongating ribosomes.

Proline-rich antimicrobial peptides (PrAMPs) are promising compounds for overcoming antibiotic resistance, one of the global health threats, and stand out from other types of AMPs by their high safety profile. The main cellular target of PrAMPs, like most modern antibiotics, is the conservative cellular structure – the ribosome. PrAMPs bind in the ribosomal tunnel, forming multiple interactions with nucleotides of 23S rRNA, and are divided into two classes depending on their mechanism of action: inhibition of elongation or termination. The N-terminal part of the peptides, which is important for the activity of class I peptides, extends into the A-site pocket, preventing the binding of aminoacyl-tRNA. A new family of PrAMPs, rumicidins, was discovered using genomic search methods. Its representatives have the longest N-terminal part, as well as a unique pair of amino acids Trp23 and Phe24 at the C-terminus. The Trp-Phe dyad forms a spacer at the constriction site of the ribosomal tunnel, stabilizing the binding and leading to increased antibacterial activity. New structural studies of the class I peptide Bac5 have demonstrated its ability to disrupt the correct positioning of the CCA-end of the P-site tRNA in the peptidyltransferase center of the ribosome, which can affect the assembly of functional initiation complexes. Class II PrAMPs, according to new data, have additional binding sites on the ribosome and have a complex effect on the bacterial cell: they disrupt the termination of protein synthesis, block the cellular ribosome release system, prevent the correct assembly of the 50S ribosomal subunits, and, possibly, affect the first stage of translocation. Recent studies expand our understanding of the antimicrobial activity of PrAMPs and contribute to the creation of future therapeutic drugs based on AMPs.

Parkinson’s disease, associated with mutations in the GBA1 gene (GBA1-PD), is the most common genetic form of Parkinson’s disease (PD), marked by clinical heterogeneity influenced by mutation type. Extracellular vesicles (EVs), key mediators of intercellular communication, are implicated in PD pathogenesis through the transport of pathological proteins and lipids. In this study, we analyzed blood plasma-derived EVs from GBA1-PD patients carrying p.N370S and p.L444P mutations and from healthy controls using cryo-electron microscopy, lipidomics, and proteomics. EVs from GBA1-PD patients were significantly larger than those from controls, with the largest size and most multilayered vesicles observed in p.N370S carriers. Lipidomic profiling identified 237 lipid species; of these, 186 lipids were altered in p.N370S and 24 in p.L444P versus controls. Mutation carriers showed distinct lipid signatures, with p.L444P samples enriched predominantly in sphingolipids, while p.N370S carriers exhibited more extensive lipid remodeling across multiple classes, including triglycerides, cholesteryl esters, and phospholipids. Notably, Cer 23:0 was elevated across all GBA1-PD groups. Proteomic analysis revealed enrichment in pathways related to lipid transport, immune regulation, and vesicle-mediated processes. Overall, GBA1-PD patients share a distinct lipidomic EV signature, with mutation-specific patterns reflecting differing mechanisms of lysosomal dysfunction. These findings support the potential of EV profiling to unravel disease heterogeneity and identify biomarkers.