Biochimica et Biophysica Acta (BBA) - General Subjects

In TMPRSS2 fusion-positive prostate cancer, ERR is involved in regulation of ERG and promotes the androgen receptor independent signaling in the cancer progression. The ERR binds to the ERREs (estrogen-related receptor response elements) present at -5042 bp of the TMPRSS2- promoter and enhances the ERG overexpression that causes prostate cancer progression. To dissect the structural basis of the ERR recognition to the TMPRSS2 promoter DNA, we have purified the full-length ERR (ERRFL), NTD deleted construct (ERR{Delta}NTD), and the DNA-binding domain (ERRDBD) proteins and performed the binding analysis with 30 bp TMPRSS2-promoter DNA (5' -AGTCCAAGGTCGGTGGATC ACAAGGTCAGG-3'). Circular dichroism analysis showed that all three ERR proteins adopt native secondary structures. DNA binding induced subtle changes in the secondary structures, while enhancing the thermal stability (Tm) of all ERRa proteins. Binding analysis showed that ERRDBD bound weakly to the DNA, whereas ERRFL and ERR{Delta}NTD exhibited substantially higher affinities ~120-fold and ~131-fold than ERRaDBD, respectively. Small-angle X-ray scattering (SAXS) analyses revealed a dimeric ERRFL structure and an ERRFL-DNA complex (2:1) structure in solution and fitted well with Alpha Fold model of apo and DNA bound complex of ERRFL. Furthermore, 100 ns dynamics simulations on apo and DNA-bound ERRa proteins showed that all proteins remained structurally stable, with flexibility largely confined to loop regions of ERRa proteins. Our biophysical, DNA binding and structural analyses have revealed the mechanism involved in ERR recognition of the TMPRSS2- promoter DNA, which provides insight into ERR-mediated transcriptional regulation and development of anticancer drugs against ERR-driven prostate cancer.

As a consequence of the overuse of conventional antibiotics, there is currently an unprecedented increase in antibiotic resistance in newer generations of pathogenic bacteria. This growing problem has led scientists to discover novel medications that could potentially reduce the usage of antibiotics, such as antimicrobial peptides (AMPs). Recent study has demonstrated that pardaxin could bind deeply into the surface of a lipid model membrane and inhibit the growth of pathogenic bacteria, including Staphylococcus aureus and Escherichia coli. In this work, the antibacterial efficacy of pardaxin was extended further by performing selective in silico substitution, driven by deep learning, Evolutionary-Scale Cambrian (ESMC) combined with conventional AMP design principles. Principal component analyses of the ESMC embeddings combined with conventional design models produced a set of single-and multiple-mutant analogues of pardaxin, which were predicted and validated with experimental data to exhibit selective antimicrobial properties against either E. coli or S. aureus, respectively. Atomistic molecular dynamics simulations further supported the notion that alpha-helical stability is a critical predictor of inner membrane activity, strongly correlated with their selective antimicrobial action tested in the lab. Overall, the findings highlighted a promising application of deep evolutionary machine learning techniques for screening a range of novel AMPs for selective antimicrobial agents.

Epigenetic regulation of chromatin dynamics via histone acetylation is one of several mechanisms by which eukaryotes regulate gene expression, DNA replication and repair, and maintain genome stability. This function is performed by histone acetyltransferases (HATs). Rtt109 is one such cytoplasmically localized HAT required for H3K56 acetylation found exclusively in fungi. Using recombinantly expressed Candida albicans Rtt109 and its chaperones, Vps75 and Asf1, we show that it can acetylate a 20-residue N-terminal H3 peptide in a coupled HAT assay only in the presence of Vps75, but not in the presence of Asf1 in vitro. This appears to be due to the fact that Rtt109-Vps75 is a high affinity stable complex, as estimated by biolayer interferometry (BLI) and gel filtration studies. The HAT activity of the Rtt109-Vps75 complex necessarily requires a flexible 118-160 residue loop of Rtt109 but not the C-terminal domain of Vps75. These results are comparable with what has been observed for the Saccharomyces cerevisiae Rtt109 homolog. In silico screening of 1,350,000 molecules from Life Chemicals Databases identified some likely inhibitors of C. albicans Rtt109 and six of them tested for binding to Rtt109 using BLI. The best ligand, F2368-0266, was used to study its effect on steady state enzyme kinetics, and found to be a competitive inhibitor of the peptide substrate but not of acetyl-CoA. Given the importance of Rtt109 in regulating virulence attributes such as hyphal morphogenesis and GPI biosynthesis in Candida albicans, and its effect on fungal pathogenesis, these results have significant clinical implications.

Glyoxal (GO) is a small, highly reactive molecule that is produced naturally in cells during normal metabolism and can also come from processed foods and oxidative stress. Because of its high reactivity, glyoxal can modify DNA and proteins to form harmful products called advanced glycation end-products (AGEs), which have been linked to diseases such as diabetes, cancer, and aging. Although glyoxal is known to modify DNA and proteins, it is not well understood whether it can form DNA-protein crosslinks (DPCs), a type of DNA damage in which proteins become permanently attached to DNA. In this study, we investigated glyoxal induced DPC formation in HeLa cells using biochemical assays and mass spectrometry-based proteomics experiments. We observed that glyoxal exposure elevated cellular DPC formation in a concentration- and time-dependent manner. Cells with reduced SPRTN expression accumulated higher levels of DPCs, suggesting that SPRTN plays an important role in repairing glyoxal induced DNA damage. Proteomics experiments revealed 469 proteins exhibited elevated DNA association in glyoxal-treated samples, including histones and other proteins involved in chromatin organization, DNA replication, DNA repair, and gene expression. In-vitro experiments confirmed that glyoxal can directly crosslink DNA with histone proteins. Overall, this study provides the first evidence that glyoxal forms DNA-protein crosslinks in human cells. These findings provide a foundation for future studies on the chemical structure, biological effects and repair of glyoxal induced DNA-protein crosslinks and their possible role in human disease.

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

Histone deacetylases (HDACs) are pivotal epigenetic regulators that modulate diverse cellular pathways by removing acetyl groups from lysine residues on both histone and non-histone proteins. Histone deacetylase 11 (HDAC11), the sole member of class IV HDACs, exhibits both deacetylation and fatty acid deacylation activities. Accumulating evidence implicates HDAC11 as a key epigenetic regulator of fundamental cellular processes, including metabolism, immune responses, and tissue development. Dysregulation of HDAC11 activity has been associated with inflammatory diseases, metabolic disorders, neurodegenerative conditions, and cancer, highlighting its potential as a therapeutic target. Although several HDAC11-specific inhibitors have been identified, none have progressed to clinical development. In this study, we aimed to discover HDAC11-selective inhibitors by integrating in silico and in vitro validation approaches. Homology modelling of the HDAC11 structure was conducted, followed by model validation, structure-based virtual screening, molecular dynamics (MD) simulations, and binding free energy calculations. We identified and validated three lead compounds and their intermediates using biochemical and cell-based assays. Fluorescence-based and HPLC-based enzymatic assays demonstrated potent inhibition of both the deacetylase and deacylase activities of HDAC11, with Inhibitor 6 and Inhibitor 3 exhibiting the strongest effects among the six compounds tested. Further, a decrease in lipid accumulation, reduced stability of the HDAC11 substrate SHMT2, as determined by immunoblot analysis and decreased cell viability, as assessed by MTT assay, confirmed HDAC11 inhibition in cellular models. The study shows that new HDAC11 inhibitors significantly reduce the viability of breast cancer cells and induce apoptosis; inhibitor 6, in particular, showed high potency, similar to the reference compound SIS-17. Flow cytometry showed that treated MDA-MB-231 cells exhibited cell-cycle arrest and increased apoptosis, a finding further confirmed by Annexin V/PI staining. Molecular analysis showed that BAX increased while BCL2 decreased, indicating that apoptotic pathways were activated in novel compound-treated MDA-MB-231 cells. The results suggest that inhibiting HDAC11 is an effective way to induce cancer cell death and provide a basis for further assessment of these compounds as potential treatments for breast cancer. Collectively, this study identifies novel zinc-chelating HDAC11 inhibitors containing a nitro-sp2 group, providing promising candidates for further therapeutic development.

The androgen receptor is a hormone-dependent transcription factor that regulates a wide range of physiological processes and plays a pivotal role in the development of prostate cancer. Its 555-residue, intrinsically disordered, N-terminal domain is involved in the modulation of transcriptional activity by recruiting co-regulators and mediating the formation of biomolecular condensates. This study reports on the characterisation of a conserved domain located in the C-terminal region of the androgen receptor N-terminal domain, at atomic level, using nuclear magnetic resonance spectroscopy. This proline rich region exhibits extensive conformational heterogeneity driven by highly populated cis proline conformers that are stabilised through interactions with adjacent aromatic residues. We demonstrate that the cis-proline population is modulated by phosphorylation as well as cancer-associated mutations. This suggests that proline driven conformational heterogeneity at the C-terminal region of androgen receptor N-terminal domain is involved in the regulatory function of this transcription factor.

Cholesterol is a key component of eukaryotic cell membranes, promoting membrane stability and modulating the function of many membrane proteins, including ion channels. In our previous work using purified human voltage-gated proton channel proteins, we showed that cholesterol inhibits the hHv1 channel by altering the conformational dynamics of its S4 segment, the key element that senses membrane voltage to control proton permeation. In the present work, we examined the effects of cholesterol analogs and potential sites in the hHv1 channel mediating cholesterol inhibition using site-directed mutagenesis and docking simulations. Our results showed that desmosterol, the immediate precursor of cholesterol, markedly attenuates cholesterol inhibition. Using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we showed that desmosterol attenuates cholesterol inhibition by promoting the intermediate and open state conformations of the S4 segment. Moreover, we identified multiple residues in the hHv1 channel that are critical for cholesterol inhibition, including Y141A in the S2 segment, which reduces cholesterol inhibition by nearly 3-fold. Our smFRET results showed that the Y141A mutation promotes the intermediate conformation in the S4 segment, which underlies the attenuation of cholesterol inhibition. Consistently, docking simulations also revealed multiple residues spanning the transmembrane domain, rather than clustered within a single localized pocket. Our work identified the key molecular determinant in the hHv1 channel that mediates cholesterol inhibition and also provided a mechanism linking the conversion between demosterol and cholesterol by DHCR24 to pH homeostasis in many cells, such as phagocytes, cardiomyocytes, neurons and microglial cells.

Membrane-bound decarboxylases couple carboxylic acid decarboxylation to the transport of Na+ ions out of prokaryotic cells. The molecular mechanism of decarboxylase action is not yet known, which contrasts with the progress achieved in studying other primary ion pumps. Measuring decarboxylase activity is complicated by slow keto-enol tautomerization of the substrates during the assay. We found that HEPES exhibits anomalously high efficiency as a general acid catalyst for C-H bond formation during the enol-to-ketone conversion of oxaloacetate. Accordingly, the addition of HEPES to the assay medium eliminated the contribution of tautomerization rate to measured decarboxylation rate. Using the dependence of oxaloacetate tautomerization rate and equilibrium on solvent properties and pH, we established that only the keto form of oxaloacetate is converted by Vibrio cholerae oxaloacetate decarboxylase. Steady-state kinetic measurements did not reveal cooperativity in oxaloacetate conversion and Na+ binding. The effects of ionophores (CCCP, valinomycin, and ETH157) on proton transport in pyranine-loaded membrane vesicles prepared from V. cholerae cells indicated that the proton required for the conversion of oxaloacetate to pyruvate is taken up from the cytoplasmic side of the membrane. Furthermore, the effects suggested that {Delta}pH generation is caused by secondary electrophoretic proton transport in exchange for Na+.These findings advance our understanding of the molecular mechanism of the decarboxylation-supported Na+ transport in bacteria.

The thermal stability of collagen triple helices is strongly influenced by the amino acid sequence of the repeating Gly-X-Y tripeptides, yet how these residue-specific interactions are integrated within an extended triple helix to determine thermal behavior remains poorly understood. Here, we addressed this question using recombinant collagen mimetic peptides (rCMPs) containing extended native sequences from the 1(I) and 2(I) chains of human type I collagen. Triple-helix formation was nucleated by a C-terminal foldon domain and further stabilized by interchain disulfide crosslinking, allowing the apparent melting temperature (T) to reflect interactions within the triple-helical domain independent of nucleation. The stabilizing effects of Pro and Y-position Arg identified in host-guest peptides were largely preserved in extended triple helices, whereas the proposed Lys-Gly-Glu (KGE) interchain salt bridge produced little measurable stabilization, demonstrating the influence of sequence context. Remarkably, identical triple-helical sequences exhibited markedly different thermal behavior when unfolding was initiated under different conditions. Nevertheless, extended triple helices differing substantially in sequence and length retained an apparently two-state thermal transition. These findings support a mechanism in which unfolding is preferentially initiated within regions of lower intrinsic stability, while the continuity of the triple helix couples neighboring regions into a cooperative unfolding process throughout the helix. This mechanism provides a plausible explanation for the longstanding paradox that extended collagen triple helices exhibit persistent sequence-dependent thermodynamic heterogeneity despite a two-state thermal transition, and a framework for investigating how sequence-dependent stability contributes to the structure and function of collagen molecules. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/741348v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f311aorg.highwire.dtl.DTLVardef@160dc76org.highwire.dtl.DTLVardef@29e989org.highwire.dtl.DTLVardef@1a3279c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Highly reactive aldehydes are generated during metabolic processes in the body, and their detoxification is essential for maintaining cellular homeostasis. Hexadecenal, a long-chain fatty aldehyde, is formed during the sphingolipid degradation pathway from the lipid mediator sphingosine-1-phosphate (S1P). However, the cytotoxicity resulting from dysregulation of hexadecenal metabolism is still unclear. To elucidate the effects of impaired hexadecenal metabolism, we analyzed the function of ALDH3A2, an aldehyde dehydrogenase in humans. Our results revealed that ALDH3A2 enzymatic activity is crucial for the suppression of DNA damage, particularly interstrand DNA crosslinks, upon S1P exposure. Furthermore, we demonstrated that hexadecenal accumulation promotes cell death accompanied by the activation of cellular stress responses and morphological abnormalities in the endoplasmic reticulum. These findings suggest that ALDH3A2 functions as a metabolic safeguard to suppress DNA damage and cell death in response to the enhanced metabolic flux of hexadecenal.

Urease is a nickel-dependent enzyme that plays an important role in urea hydrolysis and in a process named as microbial-induced calcium carbonate precipitation (MICP), which is widely used in sustainable environmental biotechnology. Despite its ecological importance, urease powers Biogrout (biocementation), a promising green technology for soil stabilization and infrastructure repair. Yet, the relationship between nickel availability, enzyme activation, and bacterial fitness remains poorly understood. In this study, we reveal a striking dual effect of nickel on Sporosarcina pasteurii: while high Ni{superscript 2} concentrations strongly inhibit growth (IC {approx} 637.7 {micro}M), they simultaneously boost specific urease activity up to six-fold. This uncoupling between biomass and enzymatic efficiency highlights a previously overlooked adaptive strategy under metal stress. Using structural bioinformatics and molecular docking, we show that Ure1--the catalytic subunit--exhibits the strongest nickel affinity (-4.3 kcal{middle dot}mol-{superscript 1}), supported by highly conserved active-site residues, whereas accessory proteins UreE and UreG display moderate and weak binding, consistent with their roles in metal delivery and GTP-dependent maturation. In addition, microscopic observations confirmed that calcium carbonate precipitation was most pronounced at intermediate nickel concentrations (approximately 400-1000 {micro}M), whereas higher concentrations ([≥]1000-1300 {micro}M) led to reduced mineral formation due to loss viable cells. Taken together, these results indicates that nickel availability controls both urease activation and bacterial fitness, and that an optimal balance is required to maximize biomenerilization efficiency in environmental applications, particularly in biocementation technology. ImportanceUrease-driven biomineralization is widely used in sustainable technologies such as soil stabilization and self-healing concrete. However, optimizing these systems requires a clear understanding of how environmental factors influence enzyme performance. This study shows that nickel, an essential cofactor for urease, plays a dual role by enhancing enzymatic activity while inhibiting bacterial growth at high concentrations. By integrating experimental data with computational analysis, we demonstrate that efficient biomineralization depends on maintaining nickel within an optimal range that balances enzyme activation and microbial viability. These findings provide practical guidance for improving biocementation processes and highlight nickel as a key regulator of urease-based environmental biotechnology applications.

Amylose contributes to starch crystallinity, but the stability of packed amylose double helices in water at elevated temperature remains insufficiently characterized. Here, we used molecular dynamics simulations to test whether chain length affects the short-timescale stability of A-type amylose oligomers in water. Six systems differing in chain length (6, 12, or 24 glucose units per chain) and oligomer size (isolated double strand or dodecamer of six double strands) were simulated, and five independent 1-s production runs were analyzed for each simulated condition. Oligomers with six glucose units showed structural collapse accompanied by increased water penetration. By contrast, dodecamers with 12 or 24 glucose units largely retained packed double-helical organization over the simulated timescale, although fraying was observed at their ends. These results indicate that chain length and lateral packing strongly affect the early structural response of amylose-like crystalline segments in hot water. The present simulations do not establish the ultimate fate of longer oligomers at longer timescales, but they identify a relative stability difference that is relevant to molecular interpretations of hydration-driven disordering in starch.

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.

This paper presents and critically reviews the results of original and some literature based experimental studies conducted by the authors last years on the structural organization of DNA in dormant (starvation stress), anabiotic dormant (4 HR treatment) E. coli cells, as well as the K12 {Delta}dps strain, which lacks the Dps protein (Dps null E. coli). The experimental data includes small-angle synchrotron radiation diffraction (SAXS) and transmission electron microscopy (TEM) data. Synchrotron radiation diffraction experiments on K12{Delta}dps cells allowed us to conclude that peaks at 44.3, 22.1, and 14.8 angstrom resolutions are associated exclusively with ordered DNA organization. Peaks at 44.3, 22.1, and 14.8 angstrom resolutions are also observed for samples of dormant (starvation stress) cells and anabiotically dormant cells. Therefore, this ordered DNA organization also applies to samples of dormant and anabiotically dormant cells. A model is proposed that considers the ordered DNA organization in the cell as a cholesteric liquid crystal. The powder diffraction pattern calculated based on this model is compared with experimental small angle X ray scattering (SAXS) data obtained on Dps-null cell samples. The model completely reproduces the key features of the experimental diffraction pattern from Dps-null cell samples. Accordingly, the cholesteric liquid crystal model corresponds to DNA packaging in dormant and anabiotically dormant cells. Cholesteric liquid crystal ordering should be further considered in all models of cellular DNA packaging. To address the question of which structural organization of DNA predominates in the cell: the cholesteric liquid crystal or nanocrystalline or whether they coexist and fully manifest themselves under different external conditions, it is necessary to utilize the latest methodological advances in structural analysis.

SMAD4 is a central transcriptional effector of the TGF-{beta} signaling pathway and a frequently inactivated tumor suppressor gene in various cancers. Missense mutations in its MH2 domain are among the most prevalent somatic alterations in colorectal cancer (CRC). These mutations are associated with disease progression and poor prognosis, yet their precise mechanistic consequences have remained incompletely characterized. Here, we show that CRC-derived SMAD4 MH2 hotspot mutations (D351H, S357P, R361C, and R361H) selectively impair co-activator recruitment without disrupting chromatin occupancy. RNA-seq profiling demonstrated broad suppression of TGF-{beta} target gene expression across all mutants. Notably, the mutations confer distinct degrees of TGF-{beta} pathway unresponsiveness: R361H is completely refractory to TGF-{beta} stimulation, whereas R361C and S357P retain partial transcriptional responsiveness suggesting allele-specific differences in the severity of co-activator interface disruption. Genome-wide chromatin binding analysis by greenCUT&RUN confirmed that all mutants maintain wild-type-like genomic occupancy, as expected given that the MH1 DNA-binding domain is intact in each case. Proximity-dependent biotinylation mass spectrometry in COLO205 cells revealed that all four mutants exhibit markedly reduced interactions with the CREBBP/EP300 histone acetyltransferase complex and BRD4 relative to wild-type SMAD4 identifying disrupted co-activator engagement. Collectively, our findings establish that SMAD4 MH2 mutations impair TGF-{beta}-induced transcription by selectively reducing CREBBP/EP300 recruitment, which provides a molecular mechanism for the loss-of-function SMAD4 phenotype in CRC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/735541v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@14f542eorg.highwire.dtl.DTLVardef@11fd220org.highwire.dtl.DTLVardef@1c3aa1org.highwire.dtl.DTLVardef@14d5a8e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Eukaryotic translation initiation is tightly regulated by interactions among translation initiation factors (eIFs) that ensure accurate start codon selection. The translation regulator, eIF5 mimic protein 1 (5MP1) contributes to this process by competing with eIF5 for binding to eIF2, thereby increasing the stringency of translation initiation. Despite its important regulatory role and emerging involvement in tumorigenesis, structural information on human 5MP1 remains limited. Here, we report the near-complete backbone and partial side-chain NMR resonance assignments of the C-terminal domain of human 5MP1 (residues 250-419), carrying a W404E substitution that disrupts dimerization. The WT protein forms a dimer at NMR concentrations, which increases the effective size of the protein and also causes disappearance of peaks corresponding to aminoacids at the dimer interface due to conformational exchange. Backbone resonance assignments were completed for 96.4% of the non-proline residues. Secondary structure was analyzed using Chemical Shift Index (CSI) and compared with the AlphaFold structural model. Regions of disagreement between the experimental and computational secondary structure assignments were further examined using 15N-NOESY-HSQC spectra, allowing experimental validation of local structural features. While the AlphaFold model accurately reproduces the overall fold of the 5MP1 C-terminal domain, several localized discrepancies were identified, particularly near the N- and C-terminal regions of the domain, where experimental NMR data support alternative secondary structure assignments. These resonance assignments and experimentally validated structural features provide a foundation for future investigations of the molecular interactions, dynamics, and functions of 5MP1 in translation initiation.

SARS-CoV, MERS-CoV, and SARS-CoV-2 exemplify the persistent threat posed by coronaviruses, with their capacity for zoonotic spill over, rapid transmission, and high mortality, and thus underscores the urgent need for broad-spectrum antiviral strategies. The nucleocapsid (N) protein, essential for RNA binding, genome packaging, and viral replication, is highly conserved among coronaviruses but remains an underexplored antiviral target. In our earlier work, we identified two small molecules, ceftazidime and sennoside A, that bind the N-terminal domain of the SARS-CoV-2 N protein and inhibit nucleic acid binding, and identified their binding sites using NMR chemical shift perturbation assays. Here, we observed that several residues involved in inhibitor binding are conserved across betacoronaviruses, suggesting a shared druggable vulnerability. We have purified recombinant N proteins from SARS-CoV, MERS-CoV, and SARS-CoV-2, and demonstrated by electrophoretic mobility shift assays that both compounds significantly reduced RNA binding. Their inhibitory concentrations (IC50) were determined using fluorescence polarization. The docking analyses indicated that both inhibitors target the RNA-binding pocket of the N-NTD, consistent with a conserved mechanism of action. Collectively, our findings reveal a conserved RNA-binding vulnerability in coronavirus N proteins and highlights the pan-coronavirus therapeutic potential of these inhibitors.

Glycosaminoglycans (GAGs) are linear, negatively charged, polysaccharides that mediate a wide variety of biologically critical interactions with proteins, underpinning growth factor signalling, extracellular matrix assembly and numerous disease processes. However, GAG-protein interactions remain under characterised, in part because of the lack of high-throughput tools to systematically profile binding across the GAG interactome. In this paper we present a novel Surface Plasmon Resonance-based array methodology utilising 16 commonly sourced GAG preparations (including chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate, heparin, hyaluronan and keratan sulphate) allowing the specificity and affinity of GAG-binding proteins to be determined. As proof of principle, we have validated the array using four established GAG-binding proteins (antithrombin III, CD44, heavy chain 1 from inter--inhibitor and Slit2), generating data consistent with the known binding specificities and quantifying affinities for many of the interactions. The array also reveals previously unreported GAG interactions, including Slit2 binding to CS and DS, and CD44 binding to chondroitin sulphate E.

Streptavidin-biotin, avidin-biotin interactions are classical models for protein-ligand binding, yet the energetic changes accompanying biotin binding remain poorly resolved. Using fluorescent dyes as energy sensors, we show that biotin binding produces two distinct regimes occurring in parallel as concentration of biotin increases cooperativity and conformational rearrangements, wherein cooperativity is observed via exchange broadening of fluorescence linewidth and conformational rearrangements exclusively observed in emission energy. Where the first biotin binding creates the highest contribution to the emission energy. Further analysis of tetramer-tetramer only interactions revealed extremely long ranged intermolecular interactions extending to hundreds of nm. The intermolecular interactions become negligible only at concentrations of approximately 10 nM for both streptavidin and avidin. Affinity values estimated for these diluted samples were below 1 nM.