Journal of Biomolecular Structure and Dynamics

BackgroundTuberculosis remains a leading global infectious killer, with BCG offering inconsistent adult protection and rising drug-resistant strains demanding novel vaccine strategies. We report the first multi-epitope vaccine construct simultaneously targeting three previously unexplored Mycobacterium tuberculosis virulence proteins; EccB3, MycP, and polyketide synthase which collectively govern nutrient acquisition, ESX secretion integrity, and innate immune evasion. MethodsUsing a reverse vaccinology pipeline, B-cell, CTL, and HTL epitopes were predicted, filtered for allergenicity, toxicity, and IFN-{gamma} induction, then assembled into an 823-residue chimeric construct incorporating beta-defensin and PADRE adjuvants with AAY/GPGPG linkers, covering [~]90% global HLA diversity. The construct underwent AlphaFold structure prediction, 3DRefine refinement, disulfide engineering, PROCHECK/ProSA validation, ClusPro 2.0 docking against TLR1/TLR2, and C-IMMSIM immune simulation. ResultsThe construct (82.3 kDa, instability index 32.48) showed strong structural quality (94.7% favoured Ramachandran residues), stable TLR1/TLR2 binding (weighted energy: -1,371.0 kcal/mol), and robust in silico immune responses and durable memory cell formation following booster simulation. ConclusionThis computationally validated construct represents a promising multi-target TB vaccine candidate warranting experimental advancement.

ObjectiveThe complement regulatory protein CD59 has been shown to promote SNARE complex assembly, yet its interaction with vesicle-associated membrane protein 2 (VAMP2) remains poorly characterized. This study aims to identify the key domain in VAMP2 that mediates the CD59 interaction and to evaluate whether CD59 point mutations affect SNARE complex assembly. MethodsThe interaction between CD59 and VAMP2 was examined by immunofluorescence confocal microscopy and co-immunoprecipitation (co-IP). The effect of CD59 on SNARE complex assembly was assessed by co-expressing CD59 with the three core SNARE proteins (syntaxin-1, SNAP-25, and VAMP2) and detecting complex formation by western blotting. Four CD59 single-point mutants were generated and evaluated in SNARE assembly assays. AlphaFold3 was employed to predict the interaction between CD59 and individual VAMP2 domains (confidence threshold: ipTM + pTM [≥] 0.75). Truncated VAMP2 constructs were further characterized by molecular dynamics simulations and co-IP. Results(1) CD59 directly bound VAMP2 and promoted SNARE complex assembly without altering individual SNARE protein levels. (2) All four CD59 single-point mutants retained the ability to promote SNARE assembly at a level comparable to wild-type CD59, despite showing differential effects on binding stability in molecular dynamics simulations. (3) The proline-rich (P-rich) N-terminal domain of VAMP2 was identified as the key binding interface; its deletion abolished the CD59 interaction, whereas deletion of the SNARE motif did not. ConclusionCD59 promotes SNARE complex assembly through interaction with the P-rich N-terminal domain of VAMP2. The examined point mutations do not impair this function, suggesting that these sites may tolerate substitutions or that redundant contact residues maintain the interaction.

The ability of small molecules to cross the blood-brain barrier (BBB) remains a major bottleneck in neurotherapeutic development. While experimental assays and machine learning approaches provide approximate permeability estimates, they lack atomistic insight into the underlying transport mechanisms. Here, we employ all-atom molecular dynamics simulations of a compositionally realistic BBB lipid bilayer to characterize the passive permeation of two bioactive propolis-derived compounds, Caffeic Acid Phenethyl Ester (CAPE) and Artepillin-C (ARC). Using steered molecular dynamics and umbrella sampling, we computed free energy profiles, diffusion coefficients, and permeability metrics across the membrane. CAPE encounters a modest barrier at the lipid headgroup region but minimal resistance within the hydrophobic core, resulting in a low free energy barrier ([~]2-3 kcal/mol) and favorable permeability (logP_eff {approx} 0.28). In contrast, ARC exhibits a substantial energetic barrier within the membrane core, leading to high resistivity and strongly unfavorable permeability (logP_eff {approx} -10.91). The heterogeneous lipid model reproduces experimentally consistent membrane properties and reveals how lipid composition modulates transport energetics. These findings provide mechanistic insight into BBB permeability and demonstrate the utility of atomistic simulations for guiding the design of neuroactive therapeutics.

Cancer is a serious public health problem, and is becoming more common, with a projected increase in deaths and more than 25 million new cases in coming decades. A number of molecular mechanisms are involved in the tumoral process, with one of them, never in mitosis A-related kinase 2 (NEK2), a serine/threonine protein kinase, being a frequent target of amplification in various malignancies that is responsible for chromosomal instability, aneuploidy and activation of several oncogenic pathways. Available kinase inhibitors are not yet optimized in terms of their pharmacokinetic properties for clinical use, and current therapies, such as chemotherapeutic agents or immunotherapies are often limited by their resistance. In silico methods represent an effective tool to search for novel potent inhibitors, before testing in animals, with time constraints and limited resources. To find new inhibitors of NEK2, we used E pharmacophore-based modeling and structure based virtual screening in this study. NEK2 was chosen as the target for therapeutic intervention and an energy optimized pharmacophore model was employed to screen the Enamine REAL library of millions of compounds. Pharmacodynamic and Pharmacokinetic properties of the Top hits were tested using ADMET profiling. These were further screened using molecular docking (standard precision and extra precision) and virtual screening to obtain three lead compounds 1, 2, and 3 which have docking score of -7.414, -8.037 and -7.562 respectively. MM-GBSA calculations were used to estimate the binding free energies for these complexes, which were determined to be -54.92, -54.18 and -49.23 kcal/mol. Lastly, 100 ns molecular dynamics simulations have been run to evaluate complex stability in dynamic situations. The overall results of the MD showed the overall stability of the NEK2-ligand complexes, and thus these three compounds are promising NEK2 inhibitor candidates and could be further validated in vitro and in vivo for clinical application. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/742111v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@14e9cfeorg.highwire.dtl.DTLVardef@24ec78org.highwire.dtl.DTLVardef@20be75org.highwire.dtl.DTLVardef@1b80c02_HPS_FORMAT_FIGEXP M_FIG C_FIG

Engulfment and Cell Motility protein 1 (ELMO1) regulates cell migration, phagocytosis, and cytoskeletal remodeling, positioning it as a compelling therapeutic target across kidney diseases, oncology, enteric infections and inflammation. Despite this potential, no approved therapeutics or clinically validated small-molecule modulators of ELMO1 currently exist. ELMO1 functions by forming a complex with DOCK180 (or DOCK2) to activate the small GTPase Rac1, and the recent structural resolution of the ELMO1/DOCK2 complex now provides an opportunity to target this protein-protein interface directly. Here, we present the first investigation into the druggability of the ELMO1/DOCK2 complex and report the initial virtual screening to identify small-molecule inhibitors of this interaction. Molecular dynamics (MD) and free energy level (FEL) studies were carried out to validate the potential of the predicted hits. This work establishes a computational framework for the development of the first generation of ELMO1-targeted therapeutics. In addition to demonstrating the drugability of ELMO1, this work introduces two open-source Python tools for the rapid analysis and visualization of protein-protein interaction and ligand-protein MD trajectories from DESMOND output files. These tools are designed to be broadly accessible, offering practical utility to the wider DESMOND user community.

Influenza A viruses H3N2 and H10N5 represent, respectively, a persistently dominant seasonal pathogen and a newly documented zoonotic threat with the latter strain variants responsible for the first confirmed human fatality in January 2024, yet no vaccine platform currently addresses co-protection against both subtypes within a unified immunogen. We report here the immunoinformatics based vaccine design and multi-layered computational validation of a 419-amino-acid multi-epitope subunit vaccine construct targeting conserved hemagglutinin (HA) and neuraminidase (NA) antigens identified through multiple sequence alignment of the avian H10N5 (A/swine/Hubei/10/2008) and H3N2 human reference strain sequences to identify viral agents undergoing mammalian adaptations. Linear B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes were predicted using ABCpred, BCEpred, BepiPred 2.0, NetMHCpan 2.1, and NetMHCpan 4.0, then filtered through VaxiJen 3.0, AllerTOP v2.1, and ToxinPred to retain only antigenic, non-allergenic, non-toxic candidates. The final construct, incorporating an avian {beta}-defensin N-terminal adjuvant with GPGPG, AAY, and EAAAK linkers, exhibited a molecular weight of 43.9 kDa, instability index of 31.15, and SOLPro solubility probability of 0.763. Tertiary structure modeling via I-TASSER and GalaxyRefine achieved 84.4% Ramachandran-favored residues. Molecular docking against TLR3 and TLR7 yielded binding free energies of -16.1 and -16.8 kcal/mol with picomolar dissociation constants. Molecular dynamics simulations confirmed complex stability over extended trajectories. Furthermore, codon optimization produced a Codon Adaptation Index of 1.0 for E. coli K12 expression. In silico immune simulation demonstrated robust activation of humoral and cellular immunity including elevated IgG1, IgM, IFN-{gamma}, IL-2, rapid NK cell expansion, and broad B-cell clonal diversity. These findings establish a computationally validated candidate capable of providing protection against influenza in multiple host organisms, warranting experimental advancement.

Human African trypanosomiasis, caused by a protozoan parasite Trypanosoma brucei, is a neglected tropical disease for which well-tolerated, conveniently administered, and highly efficacious medicines are still missing. Previously, T. brucei Phosphofructokinase was targeted by small-molecule inhibitor development efforts. This approach has shown promise both in vitro and in vivo. In this study, we have used these wet-lab results, evaluated the compounds already characterised by Molecular Dynamics simulations, found relationships between in silico and wet-lab data and used these observations to evaluate compounds that we selected through several different approaches of virtual screens. We observed that inhibitor-ATP interactions are highly predictive of the inhibitory activity. Several compounds selected through virtual screens have outperformed previously characterised compounds.

Numerous advances in quantum and computational chemistry over the last decades, well as the development of computer science, allowed utilisation of more precise and complex models, which can be now applied to much bigger systems than in the past. The authors used Gaussian, coupled with theoretical methods, to predict a new way of peptide bond formation, which could have taken place in prebiotic conditions. To better tackle this difficult task, the properties of substrates (glycine-derived radicals) were extensively analysed, using the aforementioned tool - Gaussian, paired with taking resonance and hybridisation into account, to better understand the stereochemistry and the very nature of processes taking place. The result is a series of reactions, which without any sophisticated catalysts and with relatively low energy thresholds ({inverted exclamation}20 kcal/mol) can lead to formation of dipeptides (and further, oligopeptides). The authors also hope, the other predicted properties of the investigated molecules can be of use to any researcher, who would like to utilise them in their experiments. Author summaryOur goal was to investigate a way first peptide bonds in prebiotic conditions could have been formed. This is an extremely important step in research into the beginning of life on Earth. We found a very promising series of reactions, which uses atomic hydrogen as its only catalyst and confirmed our expectations with theoretical calculations, using Gaussian. There are two radicals derived from glycine, which perform major roles in the process, so we investigated their properties with Gaussian and verified that the results are in agreement with our own theoretical considerations. This involved checking for possible geometric isomers and conformers and creating models which could explain their properties. We are well aware that such calculations have limitations and there is no model, which is 100% accurate, so our results should be further confirmed by empirical data in the future. However, we still to be as thorough as possible in how we approached the subject.

Production of recombinant proteins frequently yields inclusion bodies that must undergo refolding to yield active protein. Here, we optimized the refolding conditions for the recombinant leucyl aminopeptidase (rPepL) from Lactocaseibacillus casei expressed in inclusion bodies from E. coli. Several chemical additives were assessed for how well they facilitated an increase in refolding efficiency. The best, 0.5 M L-arginine, yielded 50.8% refolding. The addition of stabilizers, such as sucrose and glycerol, with L-arginine further increased yields to 85%. Urea at lower concentrations (0.25-0.5 M) also facilitated an increase in the refolding yield when co-added with L-arginine, whereas guanidinium chloride inhibited it. Sugars and polyols exhibited dose-dependent effects, with ranges for optima also defined. Fluorescence spectroscopy verified enhancements in the refolding under the optimized conditions. Molecular dynamics simulation under mixed solvent conditions provided atomic insights about stabilizing interactions that are likely to facilitate increased refolding. The results show that a series of aggregation suppressors and protein stabilizers can, in a collaborative way, increase the refolding efficiency for the recombinant proteins from the inclusion bodies. The protocol with the optimization using the additives L-arginine, sucrose, and glycerol is an efficient method for the production of active rPepL. This article outlines the best refolding method to recover recombinant leucyl aminopeptidase from inclusion bodies of E. coli using L-arginine combined with sucrose and glycerol. The combined experimental observations and computational simulations elucidate the molecular process of additive-induced stabilization, which elucidates how aggregation inhibition and hydrogen-bonded stabilization act synergistically. The results presented herein answer both mechanistic understanding and experimental guidance for improving protein refolding.

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 M to 200 M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health.

Structure-based drug discovery is known to apply computational methods in a tiered hierarchy, with each layer narrowing the candidate set and refining the binding picture before committing to the next, more expensive step. We present a four-tiered computational benchmarking study evaluating five engines against a panel of 36 compounds targeting B-secretase 1 (BACE1), a validated Alzheimer's disease target with extensive co-crystal ground truth. This study evaluates Flexible Docking and Boltz2 Cofolding as the primary tier, followed by Ensemble Docking, and then Protein-Ligand MD with MM/PBSA and MM/GBSA post-processing. This is then concluded with Relative Binding Free Energy Perturbation (RevFEP) as the terminal refinement layer. Each method was benchmarked against the experimental binding free energies derived from the co-crystal structures spanning -7.85 to -11.35 kcal/mol. Our findings revealed that Flexible Docking reproduced the co-crystal binding mode for 35 of 36 ligands (97.2% within 2.0 A RMSD) but did not rank potency at this resolution. Boltz2 CoFolding provided an orthogonal structural cross-check with a receptor backbone RMSD of 0.293 A against the experimental co-crystal structure. Ensemble Docking identified the optimal receptor conformation for downstream FEP setup. MD with MM/GBSA decomposition identified van der Waals complementarity as the primary potency driver (Pearson r = +0.855, R2 = 0.732 on a 10-compound subset). RevFEP delivered the highest affinity correlation of any method (Pearson r = +0.662, R2 = 0.438, Spearman p = +0.624, mean absolute error 1.02 kcal/mol across all 36 ligands), resolving potency differences within a narrow 3.5 kcal/mol congeneric window that no other engine could discriminate. We characterize what each engine contributes independently and where RevFEP delivers signals no other engine achieves.

Significance Missense mutations can lead to pathological effects in human cells. Predictive methods that account for structural context, such as AlphaMissense, can provide pathogenicity scores. The accumulation of pathogenicity hotspots can reveal important structural features within individual proteins of protein families, such as GLUT transporters. Mapping pathogenicity scores onto the structure can thus provide a mechanistic explanation of the protein function necessary for its role in the cell. Abstract Non-synonymous amino acid substitutions (missense mutations) are common in the general population; some are causative of serious disease. Depending on their structural context, they can disrupt protein function, folding, or dynamics. Computational predictive methods developed in recent years, such as AlphaMissense, provide new insights into how missense mutations affect protein structure by predicting and mapping their pathogenicity across each amino acid in the human proteome. In this study, we identify recurring patterns of pathogenicity prediction across the GLUT family membrane transporters encoded by genes slc2a1-14. Within the GLUT transporter family, we observe higher pathogenicity profiles in the transmembrane domains, particularly in pore-lining and binding-site residues. Predicted missense pathogenicity is elevated throughout residues assigned to the central cavity, suggesting sensitivity of the transport pathway. Another finding shows higher pathogenicity in specific transmembrane helices of the protein, with the same pattern across all proteins. On the other hand, we observed lower pathogenicity values in some representatives of the GLUT family. These findings show that the pathogenicity of glucose transport within the GLUT family may be shaped by functional redundancy and physiological essentiality across GLUT groups.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

Group A Streptococcus (GAS), or Streptococcus pyogenes is almost exclusive and greatly adapted human pathogen. It causes a wide array of clinical symptoms, ranging from minor infections of the skin and soft tissues to pharyngitis, meningitis, pneumonia, bacteraemia, cellulitis, puerperal sepsis, and necrotising fasciitis. The risk of S. pyogenes infection is known to be influenced by several host characteristics, including age, underlying diseases like diabetes, varicella, or skin lesions, both chronic and acute, and certain risk behaviours such as use of drugs. Household size and overcrowding are two environmental factors that significantly affect the transmission of S. pyogenes. The majority of cases occur spontaneously in the community, and preventative opportunities are still limited. A large portion of GAS-related mortality is found in low-income areas and communities. Based on aforementioned public health risk, the creation of effective therapeutic vaccines would be an excellent addition to current control measures. The purpose of this work is to address the need for new instruments to aid in the elimination of S. pyogenes infections. The discovery of high antigenic regions in several highly conserved proteins brings us one step closer to developing peptide vaccines capable of influencing the different phases of S. pyogenes infection, providing more effective defence and greater serotype coverage. This study used various techniques of immunoinformatics to design an effective multi-epitope vaccine that produced neutralising antibodies against multiple strains of S. pyogenes.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

Autophagic clearance of hyperphosphorylated tau is impaired in tauopathies, leading to the progressive accumulation of toxic tau species. Fungal metabolites provide a rich yet largely untapped source of neuroactive molecules with therapeutic potential. Here, we investigated metabolites from Lions Mane (Hericium erinaceus), Magic Mushrooms (Psilocybe spp.), and Ergot fungi (Claviceps spp.) using a computational drug-discovery workflow. We characterised their structural diversity, predicted blood-brain barrier permeability and toxicological properties, and integrated network pharmacology with protein-protein interaction and functional enrichment analyses to identify autophagy-related targets. Peroxisome proliferator-activated receptor gamma (PPARG), glycogen synthase kinase-3 beta (GSK3B), and casein kinase 2 alpha 1 (CSNK2A1) emerged as the three most promising candidates, given their complementary roles linking autophagy and tau pathology and their attractiveness as targets for multi-target drug discovery. Their interactions with fungal metabolites were evaluated by molecular docking, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring, molecular dynamics simulations, complemented by machine-learning quantitative structure-activity relationship (QSAR) modelling as an additional, ligand-based line of evidence. Molecular dynamics and MM/GBSA analyses confirmed stable, target-specific binding for Corallocin A and Erinacerin M (PPARG), Chaetopyranin and Ergocryptine (GSK3B), and Hericioic Acid D and Isohericerin (CSNK2A1), alongside Emodin, a reference compound with previously reported activity against all three targets. QSAR predictions were informative primarily for the PPARG candidates, which fell within the models applicability domain; predictions for the remaining candidates fell outside their respective models applicability domains and were therefore not interpretable as evidence for or against their prioritisation. Reanalysis of an independent hippocampal proteomic dataset from Alzheimers disease patients showed CSNK2A1 protein levels to be significantly altered in the CA3 subfield, providing an additional, correlative line of support for this target; PPARG and GSK3B showed no significant changes in protein abundance, which does not preclude their functional involvement given their extensive post-translational regulation. Overall, these findings identify fungal metabolites as promising multi-target autophagy modulator candidates and provide a systematic computational strategy for prioritising them for experimental validation in tauopathies.

Valproic acid/sodium valproate (VPA) is a widely prescribed anticonvulsant and has also been used against certain tumor cells. It is a potent modulator of gene expression. Its ability to induce apoptosis has been well documented in HeLa cells. However, another form of cell death - mitotic catastrophe - has not yet been explored in VPA-treated HeLa cells. Here, we investigated the effects of VPA treatment on mitotic catastrophe characteristics, including morphological features and their frequencies, fluorescence intensity signals of caspase-2 and p53, and the expression and abundance of DNMT1 and DNMT3B. An increased frequency of mitotic catastrophe was observed not only morphologically, but also through enhanced induction of caspase-2, involvement of p53, at least under more drastic VPA treatment, but without a decrease in DNMT1 or DNMT3B levels. Additionally, enhancement of mitotic catastrophe coincided with a reduction in mitotic chromosome abnormalities. Increased DNMT3B expression following VPA action, may be favored by previously reported chromatin decondensation induced by this drug. Enhanced CpG methylation of specific DNA sites could thus be promoted. In conclusion, VPA was shown to trigger metabolic pathways linked to different forms of cell death in HeLa cells, supporting its oncosuppressive potential.

BackgroundHelicobacter pylori coloniz the gastric mucosa of nearly half of the global population and is classified as a Group I carcinogen by the World Health Organization due to its strong association with gastric cancer. The growing prevalence of antibiotic-resistant H. pylori strains significantly compromises current therapeutic strategies, emphasizing the urgent need for effective prophylactic approaches. Research design and methodsIn this study, a novel multi-epitope vaccine was designed targeting H. pylori, incorporating epitopes from four key virulence proteins: BabB, SabB, SabA, and VacA. Using an immunoinformatics-guided structural vaccinology approach, B- and T-cell epitopes were predicted, prioritized based on immunogenicity, conservation, population coverage, and non-homology to human proteins, and assembled into the final vaccine construct. To enhance immunogenicity and specifically stimulate mucosal immune responses, the cholera toxin B subunit (CTB) was fused at the N-terminal via an EAAAK linker, a novel application in H. pylori multi-epitope vaccines. The PADRE universal epitope and additional linkers were incorporated to optimize epitope presentation and helper T-cell activation. ResultsComprehensive evaluations of physicochemical, antigenic, allergenic, and toxic properties were conducted, followed by secondary and tertiary structure modeling, refinement, and validation. Conformational B-cell epitopes were mapped, and molecular docking, binding affinity analysis, energy minimization, and molecular dynamics simulations confirmed structural stability and re-ceptor interactions. Codon optimization and in silico cloning predicted efficient expression in Escherichia coli, while immune simulations suggested robust humoral and cellular responses. ConclusionsThis study presents a promising multi-epitope vaccine candidate against H. pylori, offering a rational framework for future experimental validation and potential clinical application.

The Epstein-Barr virus (EBV) is a highly prevalent virus worldwide that is associated with several lymphoid and epithelial malignancies. However, extensive research on EBV integral membrane proteins BILF1, LMP1 and LMP2, has been scarce due to their hydrophobic transmembrane domains. Our study applies the QTY code (glutamine, threonine, tyrosine) to design water-soluble analogs of BILF1, LMP1 and LMP2 with reduced hydrophobicity, where we systematically replaced hydrophobic amino acid residues leucine (L), isoleucine (I), valine (V), and phenylalanine (F) with structurally similar polar residues glutamine (Q), threonine (T), and tyrosine (Y). We retrieved their native sequences from UniProt, identified transmembrane domains using Protter, then performed QTY design through the Protein Solubilizing Server (PSS). We then predicted native and QTY structures using in silico prediction tools AlphaFold3, ColabFold, and Boltz-2. Our analyses demonstrate that despite significant protein sequence replacements in their transmembrane domains (54.15%-61.59%) and increased intrinsic solubility, the QTY analogs exhibited minimal changes in isoelectric point (0.00-0.15 decrease) and molecular weight (0.7-1.2 kDa increase). Additionally, structural superpositions between QTY analogs and native structures using PyMOL yield low RMSD values (0.217[A] -1.202[A]). Our results demonstrate the QTY codes ability to design detergent-free analogs of BILF1, LMP1 and LMP2 with substantially reduced hydrophobicity and aggregation propensity whilst preserving native-like structures. Our results may facilitate protein characterization studies, therapeutic research on EBV, and other protocols that typically require protein solubilization.

Multiple-drug resistant (MDR) Staphylococcus aureus strains (like methicillin-resistant S. aureus or MRSA) uses an arsenal of antioxidant enzymes to mitigate host-induced oxidative stress. Among them the non-canonical Staphylococcal glutathione peroxidase (SaGpx) plays a crucial role in bacterial redox homeostasis by reducing peroxides via thioredoxin-dependent pathways. Thus, enabling oxidative stress mitigation during host infection. Despite its importance in S. aureus, its role in bacterial pathogenesis remains unexplored. This study aimed to elucidate the possible role of SaGpx in Staphylococcal virulence. First, we determined the high-resolution crystal structure of SaGpx (at 1.65 [A] resolution) using X-ray crystallography. Guided by the catalytic cleft architecture of SaGpx, small-molecule based inhibitors were then rationally designed and synthesized. These inhibitors exhibited good binding affinity to SaGpx and complete enzymatic blockade. These inhibitors exhibited potent anti-S. aureus activity (MICs 6.25-31.25 M) along with no cytotoxicity in L929 fibroblast wound-healing assays. Furthermore, the in vivo antibacterial ability of these inhibitors was evaluated using S. aureus-infected skin wound mouse model, where these compounds show potent antibacterial and wound healing ability supported by subsequent histological as well as immunohistochemical analysis. These findings suggest SaGpx as a possible virulence determinant in S. aureus and position these synthesized inhibitors as promising antivirulence therapeutics. HighlightsO_LIThe high-resolution crystal structure of Staphylococcal glutathione peroxidase is solved. C_LIO_LIBased on the SaGpx catalytic site, ,{beta}-unsaturated ketoesters derivatives are synthesized. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives inhibit SaGpx activity and binds the protein at M range. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives show in vitro antibacterial activity against S. aureus at low M range. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives show in vivo antibacterial and wound healing ability S. aureus-infected skin wound mouse model. C_LI