Biochimica et Biophysica Acta (BBA) - General Subjects

Proteases are an important class of proteolytic enzymes having great importance in both basic science and industrial applications. While cells tightly regulate the spatio-temporal activity of different proteases for cellular homeostasis, mis-regulation often leads to adverse effects. In this context, the protease activity of papain and its activation by L-cysteine is poorly understood in the literature. Herein, we discover that the protease activity of papain can be effectively regulated via a spontaneous liquid-liquid phase separation (LLPS) pathway. We show that papain undergoes biomolecular condensation via spontaneous LLPS under macromolecular crowding through the involvement of intermolecular hydrophobic interactions. Secondary structure analyses revealed a compact conformation of phase-separated papain with increased -helix content. Although native free papain is found to be active towards synthetic and protein substrates, the proteolytic digestion produces heterogeneous peptide aggregates. In contrast, we found that papain droplets remain dormant toward protein digestion due to the disulfide linkage of the active cysteine residue (Cys-25) in its compact conformational state. More importantly, we show that the protease activity of phase-separated papain can be reactivated in the presence of L-cysteine to produce uniform soluble peptide fragments. Our findings indicate that although disulfide linkages are not necessary for the phase separation of papain, upon phase separation, intermolecular interactions between phase-separated papain result in the formation of disulfide linkages involving active Cys-25 residues. The present discovery has tremendous technological importance to boost the efficacy of meat tenderization in the food industry.

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.

Glioblastoma multiforme (GBM) is among the most aggressive malignant brain tumors originating from glial cells and characterized by severe infiltration into surrounding brain tissue, rendering early detection difficult with current diagnostic imaging methods. S100A4 has been identified as a biomarker protein associated with glioblastoma invasiveness due to its role in cell motility and tumor metastasis. Similarly, midkine (MDK) poses an optimal biomedical target for identifying GBM invasive phenotypes because of its connection to the tumor microenvironment and infiltrative proliferation. Both proteins notably possess a positive charge that interacts electrostatically with the negatively charged phosphate backbone of DNA. It has been established that early molecular detection remains a critical unmet need. This study investigates a promising strategy for GBM diagnosis based on how S100A4 and MDK can selectively bind with DNA tweezer nanostructures. Computationally predicting eight distinct nucleotide sequences yielded three-stranded, hinge-scaffolded tweezer conformations for each candidate. The target protein and DNA structures, derived from AlphaFold, were paired together by molecular docking simulations conducted with HDOCK. Docking analyses evaluated binding affinity, structural complementarity, and conformational stability of the complexes formed. Among the evaluated candidates, DT3_8 computationally established the most biochemically robust interaction with both biomarker proteins. Selectivity is especially important because many S100 proteins share similar electrostatic profiles, yet DT3_8 indicates stronger selectivity for S100A4 and MDK over other S100 family proteins. These findings establish a biomechanical basis for the development of nanoscale DNA biosensors, which suggests the potential for detecting invasive GBM phenotypes, preceding radiographic manifestation and pending experimental validation.

Cholera, caused by Vibrio cholerae, continues to pose a serious global public health challenge, with its impact worsened by rising antibiotic resistance associated with bacterial biofilm formation. This study reveals the role of the hypothetical protein (HP) TYC33605.1 in cyclic-di-GMP (c-di-GMP)-mediated biofilm regulation and identifies natural inhibitors that disrupt this mechanism. Functional annotation revealed TYC33605.1 as a membrane-associated diguanylate cyclase (DGC) with GGDEF and sensory domains, critical for c-di-GMP synthesis and biofilm persistence. Homology modelling and molecular dynamics (MD) simulations validated its stable 3D structure (C-score: -1.22, Ramachandran favoured regions: 91.1%) and dynamic behaviour (average RMSD: 8.55 [A]). Virtual molecular docking screening of 1,092 natural compounds identified Luteolin (CID 5280445) and Sativanone (CID 13886678) as top candidates, exhibiting strong binding affinities (-9.1 and -9.0 kcal/mol, respectively) and forming hydrogen bonds, {pi}-cation, and hydrophobic interactions with key residues (Glu293, Arg364, Ala176). MD simulations (100 ns) confirmed complex stability, with Luteolin and Sativanone showing lower RMSD fluctuations (7.78 [A] and 8.13 [A]) compared to the control and apoprotein. The ADME/Tox profiles highlighted favourable pharmacodynamics (PD), pharmacokinetics (PK), high gastrointestinal absorption, no hepatotoxicity, and drug-likeness (Lipinski compliance). Principal component, probability density function, and binding free energy analyses underscore ligand-induced conformational stability. This study proposes the molecular characterisation of the HP and the bioactive compounds Luteolin and Sativanone as promising inhibitors targeting TYC33605.1, offering a novel strategy to combat biofilm-mediated antibiotic resistance and a framework for analogous antimicrobial discovery in Vibrio cholerae.

Reactive oxygen species (ROS) are central signaling molecules in many biological processes by inducing oxidative modifications of protein cysteine residues, including S-glutathionylation. Increasing evidence supports that ROS contribute to cancer progression via promoting cancer cell migration, invasion, and metastasis. Nevertheless, the protein targets of S-glutathionylation that regulate cancer cell motility remain ill-defined. In this study, we report on the redox regulation of ARHGEF7, a guanine nucleotide exchange factor highly expressed in metastatic cancer cells, that plays a major role in regulating cell migration. Our data demonstrates that ARHGEF7 is selectively glutathionylated at the highly conserved C312 residue in its PH domain, which is implicated in regulating its enzymatic activity. Breast cancer cell lines showed increased cell migration and invasion upon glutathionylation of ARHGEF7 at C312 in response to both oxidative stress and epidermal growth factor (EGF). Mechanistically, upon C312 glutathionylation, ARHGEF7 exhibited significantly enhanced binding to Rac1 and increased Rac1 recruitment to the cell membrane and lamellipodia. ARHGEF7 S-glutathionylation also increased its enzymatic rate of GDP-GTP nucleotide exchange, resulting in Rac1 activation. Consequently, ARHGEF7 C312 S-glutathionylation induced Rac1-PAK1 activation and their downstream pathways, including LIMK1 and MEK1, thereby enhancing migration and invasion. Our data reveal a new redox player in cell migration, with its potential implications for ROS-induced cancer progression.

Burkholderia mallei, a facultative intracellular Gram-negative pathogen, is the causative agent of glanders that primarily affects solipeds and sporadically transmitted to humans. Current interventions mainly rely on antibiotics; however, increasing resistance and the lack of a licensed vaccine further complicate disease management. In the present study, a consensus-based computational framework was employed on the B. mallei turkey2 proteome. Total 59 proteins - including porins, TonB receptors, autotransporters, and efflux components - were identified as surface exposed outer membrane {beta}-barrel (OMBB) proteins that were used to design a multi-epitope vaccine (MEV) construct. B- and T-cell epitopes were predicted from 59 proteins, and ten epitopes each of cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell were chosen based on their antigenicity, non-allergenicity, non-toxicity, surface accessibility, and conservation across 32 B. mallei strains. The MEV was included with suitable adjuvants at the N-terminus to enhance its immunogenicity. The 780 amino acid MEV construct was predicted to be antigenic, and soluble upon overexpression with 62.69% random coils, while the rest formed -helices and {beta}-strands. The tertiary structure of the MEV was generated and subsequently validated, indicating good structural quality. Molecular docking of the MEV with toll-like receptor 4 (TLR4) demonstrated strong affinity, and molecular dynamics simulation confirmed the structural stability of the MEV-TLR4 complex. In-silico immune simulation showed the capability of MEV to induce a strong immune response. The study proposes an MEV construct by utilizing surface exposed OMBB proteins which directly interact with the host and serve as effective immunogenic targets against B. mallei infection. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/727591v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@10cd6d8org.highwire.dtl.DTLVardef@1ed3f0borg.highwire.dtl.DTLVardef@c6173forg.highwire.dtl.DTLVardef@1204f73_HPS_FORMAT_FIGEXP M_FIG C_FIG

Up to 50% of adults with colorectal cancer (CRC) are at risk of progressive involuntary loss of skeletal muscle mass and function known as cachexia. Available options to prevent and treat cachexia in cancer survivors are currently limited. Long-chain polyunsaturated fatty acids (LC-PUFAs) and their bioactive metabolites, termed specialized pro-resolving lipid mediators (SPMs), promote the resolution of inflammation and support muscle growth and repair. However, prior studies of cachexia have mainly focused on fish oil supplements, and it is not fully understood how different individual omega-3 (n-3) and omega-6 (n-6) LC-PUFAs mediate CRC-induced muscle wasting. In addition, the crosstalk between cancer cells, the host immune system, and skeletal muscle cells in response to LC-PUFA treatments remains unclear. This study aimed to examine the effects of n-3 and n-6 LC- PUFAs on CRC-induced muscle wasting and the underlying cellular and molecular mechanisms involved. Using murine C2C12 skeletal muscle cells and CT26 colorectal carcinoma cells, we investigated the impacts of LC- PUFAs including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3), and docosahexaenoic acid (DHA, 22:6n-3) on CT26-induced muscle cell wasting in the presence or absence of lipoxygenase (LOX) inhibitors such as NDGA or BLX-3887. We also examined the lipidomic profile of C2C12-CT26 co-cultures in response to individual LC-PUFA treatments. Our results suggest that LC- PUFAs including ARA, EPA, DHA, and DPA each individually protect against CRC-induced muscle cell wasting in vitro, and these protective effects are dependent on 15-LOX activity. Furthermore, we found that C2C12-CT26 co-culture produced mature SPMs in response to individual PUFA treatments. Taken together, this study suggests that individual n-3 and n-6 LC-PUFAs can mitigate CRC-associated cachexia primarily by producing 15-LOX- derived bioactive lipid mediators.

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.

Combatting biofilm-associated methicillin-resistant Staphylococcus aureus (MRSA) infections remains a formidable challenge, largely due to bacterias heightened resistance to antibiotics and evasion of the hosts immune responses. Inhibiting biofilm formation or promoting biofilm dissolution is believed to disrupt the protective matrices and expose bacterial cells to immune clearance or antimicrobial treatments. Cross- helical amyloid fibrils of phenol-soluble modulins are the key structural components of the MRSA biofilm. In this study, we present a novel retro-inverso peptide that successfully inhibits cross- amyloid formation, induces disassembly of pre-formed fibrils of the biofilm-forming modulin peptide, and efficiently disperses MRSA biofilm biomass in a dose-dependent manner. The biophysical studies of the interaction of the designed peptide and modulins have suggested a mechanism for the biofilm disruption. The findings highlight the potential of the proteolytically resistant mirror-image peptide as a promising therapeutic agent for the treatment of biofilm-associated MRSA infections.

Heat shock protein 90 (HSP90) assists protein folding and maturation of many important signaling proteins. In various diseases, HSP90 clients contribute to aberrant signaling, and HSP90 inhibition is being explored as a potential therapeutic approach. Commonly researched HSP90 inhibitors target the ATP-binding pocket, thereby disrupting the ATP-induced closure of HSP90. Drugs disrupting the HSP90 ATPase cycle by targeting the closed state of the chaperone have not been developed. Here, we present de novo design and selection of protein binders interacting exclusively with the closed state HSP90. Two such binders, H2 and H4, were identified that feature a similar fold and comparable affinities for HSP90 but display different binding kinetics. The structures of the HSP90 complexes with H2 and H4 were determined by cryo-EM single particle analysis, and they revealed high accuracy of the BindCraft predictions. H2 and H4 compete with p23 at one but not both symmetrical p23 binding sites on HSP90. H2 expressed in HEK293T cells moderately elevated expression of HSP70 and had no effect on the HSP90 level, suggesting muted heat shock response. Overall, this study demonstrates that the de novo binders represent novel and promising tools to probe the potential utility of HSP90 inhibition by targeting its closed state.

Mechanistic elucidation and inhibition of the pathogenic aberrant mitochondrial localization of the RNA/DNA-binding protein, TDP-43, can help in the therapeutics of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A mitochondrial localization sequence of TDP-43, M1, is largely solvent inaccessible, therefore, how it interacts with the mitochondrial import machinery to facilitate TDP-43s transit to mitochondria is unclear. Towards this, we examined the unfolding TDP-43s N-terminal domain (NTD) that hosts M1, using equilibrium all-atom molecular dynamics (MD) simulations, and observed an early loss of the hydrogen-bonded interactions between {beta}4-{beta}5 bridge and the interactions involving residues Phe-35 and Gly-40 of M1, indicating structural lability of M1 to become solvent-accessible that may enhance its interaction with the mitochondrial receptor(s) for import. Furthermore, via virtual screening of 2,115 FDA-approved and 515,545 non-FDA-approved small molecules from ZINC15 database towards binding to M1 and inhibiting TDP-43s mitochondrial import, we identified a molecule, ZINC73240059, that was previously characterized as an inhibitor of MAP kinase-activating protein kinase 2 (MAPKAPK2). ZINC73240059 remains stably bound to M1 of NTD during MD simulations manifesting negative Gibbs free energy ({Delta}G) with significant contribution from Pro-36 of M1. Overall, ZINC73240059 can be a molecule of interest towards thwarting TDP-43s pathogenic mitochondrial localization in ALS.

Nuclear organization is dynamic and originates from the fundamental subunit of chromatin, the nucleosome. Post-translational modification of nucleosomal histones, particularly within intrinsically disordered histone tail regions, provides a dynamic regulatory mechanism of accessibility for chromatin-templated processes. While the epigenomic impacts of lysine acetylation and serine phosphorylation in the histone H3 tail are well-known, how these charge-altering post-translational modifications (PTMs) alter nucleosomal tail conformational dynamics remains incompletely characterized. Given that the functional implications of these PTMs are, at least in part, a consequence of modified nucleosome conformation, systematically cataloging the impact of histone PTMs on nucleosome dynamics provides crucial insight into both baseline cellular activity and epigenetic dysregulation that occurs in disease. Previously, our lab demonstrated that arginine citrullination mimetics lead to regional increases in H3 tail dynamics within nucleosome core particles. Here, we performed nuclear magnetic resonance spin relaxation experiments to investigate the effects of lysine acetylation and serine phosphorylation on H3 tail picosecond-nanosecond (ps-ns) dynamics. Using lysine-to-glutamine and serine-to-glutamate mutations as acetyllysine and phosphoserine mimetics, respectively, we found that these PTMs increase ps-ns conformational dynamics regionally around the PTM site, with a position-dependent effect. Additionally, we show that the type of PTM influences the extent of these increases: in general, the effect of mimetics trends in the order of phosphorylation [&le;] acetylation < citrullination, suggesting a tunable method for altering histone tail dynamics. Taken together, these results illustrate the role of nucleosome conformational dynamics in conveying the effects of epigenomic PTMs, elucidating a mechanism of the histone language.

The Na+/K+-ATPase (NKA) regulates ion balance in the kidney and influences cellular processes like proliferation and apoptosis through its signal transduction. The endogenous ligand 20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to inflammation and fibrosis in chronic kidney disease (CKD) and inhibits NKA activity in renal tubules. However, the molecular mechanism of this interaction remains unclear. In this study, we used in-silico approach to investigate the potential interaction between 20-HETE and NKA. Various ligands, including known NKA ligands such as cardiotonic steroids (CTS), 20-HETE, and negative controls, were docked using rigid and Induced Fit Docking to predict the affinity of the ligands toward NKA. Binding free energy calculations with the Prime Molecular mechanics with generalized Born and surface area (Prime MM/GBSA) tools were used to confirm the involvement of key amino acids in ligand-receptor interactions. The docking analyses revealed that 20-HETE exhibited a binding affinity comparable to negative control, with some differences between rigid and induced fit docking. Binding free energy data highlighted key amino acids in the 20-HETE and NKA interaction. Interaction fingerprint and mutations such as Ala330Gly and Val329Ala significantly reduced binding free energy, while Thr804Ala showed a notable decrease, underscoring the potential importance of these amino acids in ligand stabilization. These findings provide computational evidence supporting potential direct interaction between 20-HETE and NKA and identify candidate residues for future experimental validation.

Glycation, a non-enzymatic reaction occurring between sugars and biological macromolecules, plays a critical role in ageing and disease pathogenesis. Methylglyoxal (MG) is a highly reactive -oxoaldehyde that leads to the formation of endogenous advanced glycation end products (AGEs). These AGEs are associated with diabetes and many other diseases, including neurodegeneration and cancer. This is often through interactions with the receptor for advanced glycation end products (RAGE). Inhibition of glycation/AGEs formation using natural products to target cancer is an area of recent interest. In vitro AGEs formation was observed by browning of samples, increased fluorescence, and carbonyl stress. MG induced changes in the structure of BSA were analysed using electrophoresis, spectroscopy, TEM, AFM, DLS, and CD spectroscopy. Our results show that AGEs form random structures, oligomeric aggregates, and {beta}-sheets. Thioflavin T and Congo red staining further validated these findings. Galangin and Caffeic acid demonstrated significant antiglycation activity, suppressing AGEs formation in vitro. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/737425v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@113b391org.highwire.dtl.DTLVardef@7208a1org.highwire.dtl.DTLVardef@94c2e1org.highwire.dtl.DTLVardef@867b85_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMethylglyoxal-induced Advanced Glycation End Products were prepared in vitro C_LIO_LIMethylglyoxal -induced structural modifications in BSA C_LIO_LIAGEs were characterised using various parameters C_LIO_LIBoth fluorescent and non-fluorescent AGEs were formed. C_LIO_LIPhytochemical treatment induced inhibition of AGEs formation C_LI

Bordetella produce a wide array of virulence factors. These factors are involved in bacterial colonization and evasion of immune defenses. Our recent studies revealed that the bacteria produce an exoglycan, Bordetella oligosaccharide (BOS). B. petrii is the evolutionary early divergent species of the genus Bordetella. This study has focused on the investigation of two B. petrii type strains: clinical and environmental. We employed nuclear magnetic resonance (NMR) analyses to elucidate the structural differences between their lipopolysaccharides. Our findings revealed that the LPS of clinical B. petrii strain comprises a hexasaccharide unit, that was structurally identical to the BOS. This form of LPS is only a minor population in the bacterial outer membrane of the environmental strain. In addition to the cell-bound BOS, its secreted glycoform was also found in growth media of B. petrii. Anti-BOS neoglycoconjugate antibodies cross-reacted with B. petrii LPS. This suggest that the newly identified BOS associated with B. petrii PS would be a potential vaccine element against Bordetella.

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

Protein homeostasis - i.e., proteostasis - is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In this work, we show that the Rqc1p protein is necessary for the thermoadaptation of S. cerevisiae to heat shock, as RQC1-deficient yeast is sensitive to elevated temperatures. In silico approaches - such as multiple sequence alignment, structural analysis, and molecular dynamics simulations - confirmed earlier predictions that Rqc1p shares characteristics with the bHLH family of proteins. We also verified, through computational prediction of sub-cellular localization, that S. cerevisiae Rqc1p contains nuclear localization signals, suggesting that this protein can potentially be translocated toward the nucleus, thereby broadening its current range of recognized biological functions in this organism. Also, analysis of yeast transcriptomes subjected to heat shock showed that Rqc1p mRNA levels do not fluctuate in response to heat shock, suggesting that cellular concentrations of Rqc1p are already at optimal levels to elicit a rapid and effective response during thermal stress in S. cerevisiae.

In recent years, aptamers have transitioned from mere laboratory tools to highly potent molecular recognition agents capable of overcoming the strict limitations of conventional antibiotic therapies. We have developed AptBacterialDB, a manually curated, large, comprehensive database of experimentally validated antibacterial aptamers spanning 1996 to 2026. The database contains a total of 2131 aptamers targeting approx 75 different bacterial classes, and 124 aptamer targets with 95 entries found in UTexas databases, 97 in AptaDB, and 28 in Aptabase. It contains 1555 unique aptamer sequences, 189 unique modifications, 40 different selection approaches, and 44 different affinity methods. It integrates detailed annotations of about 20 fields, including sequence information, nucleic acid type, binding affinity, modifications, experimental and functional details. The secondary structure of the aptamers was predicted using ViennaRNA Package 2.0, demonstrating that they adopt mostly stable conformations, with a structured stem region. MySQL was implemented for database development, and a knowledge graph was integrated using ArcadeDB/openCypher for graphical visualization of aptamer-target-organisation relationships. Facilities such as different search modes, browsing, similarity search, REST API access, and entries linked to the existing database for a broader view of the aptamers have been provided. AptBacterialDB (https://webs.iiitd.edu.in/raghava/aptbacterialdb/) provides a user-friendly centralized platform to accelerate antibacterial aptamer research, therapeutic development, biosensor design, and computational modelling efforts.

Multi-functionality in extant enzymes, including the ability to transform multiple substrates, is thought to arise, in part, from conformational flexibility. The hexokinase protein family represents a classic model system for investigating the relationship between substrate specificity and conformational change. Within this family, human glucokinase (hGCK) displays notable degrees of conformational heterogeneity, including an intrinsically disordered loop. The extent to which these structural features contribute to the breadth of hGCKs substrate scope is unknown. Here, we investigate the substrate specificities of extant and ancestral glucokinases that span the evolutionary emergence of conformational heterogeneity in this family. We show that extant hGCK catalyzes the ATP-dependent phosphorylation of glucose, 2-deoxyglucose, mannose, glucosamine, fructose, allose and galactose with catalytic efficiencies ranging from 6.3 x 103 M-1 sec-1 to 0.33 M-1sec-1. A glucokinase ancestor from early vertebrate evolution (vGCK), which also displays conformational heterogeneity and disorder, phosphorylates these same seven substrates with similar kcat/Km values. An antecedent, chordate glucokinase (cGCK), which displays reduced conformational heterogeneity and lacks intrinsic disorder, also transforms these same substrates, but with higher overall catalytic efficiencies and markedly lower Km values. Notably, however, the ratios of kcat/Km values for individual substrate pairs, which define specificity, are unchanged for all three enzymes. Our results demonstrate that substrate specificity is not correlated with conformational diversity in GCKs and support a model in which the differences in catalytic efficiencies of various substrates arise from differences in the ability to form the ground state enzyme-carbohydrate binary complex.

Genomic DNA is subject to forces and torsion. Some arise mechanically, while others can be entropic, such as those due to crowding within the nuclear environment. Indeed, about 30-40% of the cell is occupied by molecules other than water, and of these, the vast majority are macromolecules. Here, we explore both experimentally and theoretically the interplay between tension, torsion, and macromolecular crowding. Using pharmaceutically relevant crowders of different molecular weights, Dextran 70, and polyethylene glycol (PEG), we observed that macromolecular crowding of unwound, stretched DNA effectively opposed the tension and promoted the formation of plectonemes. A theoretical model representing the equilibrium between B- and L-form DNA fit to the experimental measurements indicates the contractile tension produced by macromolecular crowding of DNA. SIGNIFICANCE STATEMENTDistinct DNA conformers are involved in different cellular processes. Genomic DNA is both stretched and unwound by enzymes in a crowded intracellular medium. This can induce conformational changes between extended, twisted and more compact, plectonemic forms. This study explores the effect of macro-molecular crowding on the conformations of DNA subject to tension and torque. Fitting experimental data to a model for the right-to-left-handed DNA transition, we show that macromolecular crowding induces a contractile force that favors DNA writhe and that such force depends both on the concentration and molecular weight of the crowder.