Journal of Biomolecular Structure and Dynamics

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.

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.

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.

Interleukin-13 (IL-13) is an immunomodulatory cell signaling cytokine that has been implicated in neurodegenerative disease and chronic inflammation. IL-13 binds to its low and high affinity receptors, IL-13 receptor 1 (IL-13R1) and IL-13 receptor 2 (IL-13R2), respectively, with residence times that vary accordingly. As the binding kinetics of the cytokine-receptor complexes influence cellular responses, we employed the molecular dynamics (MD) simulation-based{tau} -random acceleration molecular dynamics method ({tau}RAMD) to compute relative residence times for wild-type (WT) IL-13 and 19 IL-13 mutants to the two receptors. Comparison with experimental kinetic data shows that the{tau} RAMD computations capture the trends in residence times. Analysis of simulated dissociation trajectories of the cytokine-receptor complexes reveals two distinct dissociation pathways of IL-13 from each of the receptors. This study thus pinpoints key determinants of the interaction of IL-13 with its receptors which could be targeted for therapeutic design. Statement of SignificanceCytokines are regulatory proteins that bind to cell surface receptors and thereby send signals to the cellular interior. Interleukin-13 (IL-13) is a cytokine that has a low and a high affinity receptor. It has important physiological roles, and its deregulation is involved in diseases such as atopic dermatitis and asthma. We employed a molecular dynamics simulation-based method to compute the effects of changes in the sequence of IL-13 on the lifetimes of complexes of IL-13 and its receptors. Comparison with experiments supports the validity of the computational approach and analysis of the simulations reveals two distinct ways in which IL-13 dissociates from each receptor. These results thus provide a map for targeting IL-13 - receptor interactions for the design of therapeutics.

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.

Per-and polyfluoroalkyl substances (PFAS) constitute a diverse class of persistent synthetic chemicals utilized across industrial, medical, and consumer sectors that are pervasive global pollutants. Exposure to PFAS is linked to adverse impacts on both innate and adaptive immune systems. Human lactoferrin (hLF) is a key antimicrobial component of the developing innate immune system present in colostrum and breast milk. We hypothesized that hLF is a potential PFAS binding protein related to PFAS immunotoxicity. The results of thermal stability experiments indicated that all 11 tested PFAS bind and destabilize the structure of hLF. Notably PFBA, PFOS, HFPO-DA, and 6:2 FTSA decreased apo-hLF melting temperatures from 64oC to [≤] 37oC, suggesting that PFAS exposures destabilize the native hLF protein under physiological conditions. Relative binding affinities (Kd) ranged from 0.2-11 mM across tested PFAS. Molecular docking was used to confirm experimental binding affinities and identify molecular interactions involved with PFAS binding. Calculated Gibbs Free Energies of binding ranged from -4.4 to -8.8 kcal/mol. Together, these results demonstrate that PFAS bind hLF at affinities comparable to human serum albumin and other PFAS binding proteins, and that some PFAS can destabilize hLF protein structure at physiologically relevant temperatures and conditions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by progressive motor neuron degeneration. Mutations in the SOD1 gene represent the second most common genetic cause of ALS (ALS), and distinct SOD1 missense variants present with markedly different clinical profiles. A4V leads to an aggressive form of the disease (median survival [~]1y), H46R confers a mild, slowly progressive course and I113T exhibits an intermediate phenotype. The molecular basis by which these mutations produce divergent clinical outcomes remains poorly understood. We performed extensive classical molecular dynamics simulations of wild-type SOD1 and the three ALS-associated variants in the apo monomeric state to attempt to investigate the mechanisms behind such phenotypic differences. Structural stability, global compactness, and conformational flexibility, as well as analysis of collective motions between residues and estimation of free energy, were assessed. The H46R, A4V, and I113T variants exhibited distinct dynamic behaviours, highlighting differences in structural stability, local flexibility, and intramolecular interactions. These findings suggest that specific structural regions may contribute differently to protein dysfunction and could represent key elements for understanding the relationship between molecular dynamic properties and the differing clinical severity associated with these variants. Most strikingly, H46R exhibited exceptional structural stability across every analytical level, the lowest global deviation, most attenuated local flexibility, strongest internal dynamic coordination, and the deepest, most confined free energy basins of any system examined. This convergent multi-layered evidence of structural restraint provides a compelling mechanistic basis for the mild and slowly progressive clinical course of H46R ALS, suggesting that enhanced conformational rigidity, rather than bulk destabilisation, is the defining biophysical feature of this variant, and that its pathogenic mechanism operates through a route fundamentally decoupled from the aggregation-driven toxicity that characterises the more aggressive SOD1-ALS mutations.

The High Mobility Group Box 1 (HMGB1) protein performs multiple essential functions in the body, ranging from DNA regulation to the activation and mediation of immune responses. However, HMGB1 has been also implicated in several pathological conditions, such as rheumatoid arthritis, sepsis, autoimmune diseases, tumors, and Alzheimer's disease. As a result, HMGB1 is of increasing interest as a therapeutic target. Binding to heparin has been reported to inhibit HMGB1's pathological activity during sepsis in clinical settings. In this work, we compare the interactions of HMGB1 with heparin and its' synthetic analog linear polyglycerol sulfate (lPGS) from the viewpoint of stability and changes to association behavior. This analysis focuses on thermal stability, secondary-structure changes, and particle-size evolution using nano-differential scanning fluorimetry (nanoDSF), circular dichroism spectroscopy (CD), and dynamic light scattering (DLS).

Vanadium (V) is a potential insulinomimetic that can modulate carbohydrate metabolism, but its biological effects are sensitive to chemical form, concentration, and sex. Chelation of vanadium with organic ligands, in particular citrate, allows to increase its bioavailability and optimize pharmacokinetic properties. The aim of the study was to evaluate tissue-, dose-, and sex-dependent changes in physiological parameters and activity of the key glycolytic enzyme - lactate dehydrogenase (LDH) - under the influence of vanadium citrate. The study was conducted on 6-week-old Wistar rats of both sexes. The animals received vanadium citrate orally for 36-38 days at doses of 3, 12.5, and 50 g VCit/kg body weight. LDH activity in skeletal muscle, liver, kidney, and pancreas was investigated. No pronounced toxic effect on physiological parameters was detected: body weight dynamics corresponded to age norms, no behavioral changes were observed. LDH activity demonstrated pronounced sexual dimorphism and depended on the dose received. It was established that the optimal dose, which provides a modulating effect without signs of metabolic stress, for females is 12.5 g VCit/kg, while for males - 3 g VCit/kg. The most significant changes in LDH activity were recorded in the pancreas at a dose of 50 g V/kg, where the indicators decreased from 0.81 to 0.31 mol/(min x mg protein) in females and from 1.02 to 0.28 mol/(min x mg protein) in males. The effect of vanadium citrate on carbohydrate metabolism, as well as its dose-, tissue- and sex-specific nature, is likely determined by a dual action: the insulin-like effect of vanadium (redirecting pyruvate to oxidation) and the allosteric inhibition of glycolysis by the citrate ligand (substrate limitation for LDH). The obtained results emphasize the importance of considering sex and dose in the research and development of metabolically active compounds.

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.

We published recently that one of the main constituents of cannabis products, cannabidiol (CBD), is an efficacious negative allosteric modulator (NAM) of the mu opioid receptor (MOR1) (Bosquez-Berger et al., 2023). Here, we investigated how the presence of cannabidiol (CBD) is associated with fentanyl (FEN) binding across MOR1 conformations. We performed molecular dynamics simulations of systems containing FEN alone or FEN+CBD in three mouse MOR1 conformational backgrounds: active-like 5C1M, inactive-like 4DKL, and a modeled Morph50 intermediate between the 5C1M and 4DKL conformations. Three independently seeded 200 ns trajectories were analyzed per model and condition (18 trajectories total), with the trajectory treated as the independent unit. Across the matched 0-200 ns window, consensus CBD contacts and CBD-associated changes in FEN contacts were strongly state dependent. Corrected intracellular TM3 to TM6 analyses separated the expected active-like, intermediate, and inactive-like backgrounds but did not identify a CBD-associated shift that was consistent across both geometric definitions and all three replicates. Equal-weight replicate-composite density maps preserved both the shared ligand distributions and this between-trajectory variability. These descriptive results support receptor-state-dependent CBD, FEN, MOR1 interactions while emphasizing the limited inferential power of three trajectories per condition.

Antibodies generally exhibit high specificity for their cognate epitopes, but structural and physicochemical similarities between distinct epitopes can enable an antibody to recognize different antigens, resulting in cross-reactivity. This property can be exploited for antibody repurposing. To identify epitopes that share such similarities, both sequence- and structure-based approaches can be employed. In this context, 3D Zernike descriptors provide a compact representation of protein surface geometry as numerical feature vectors, enabling quantitative comparisons independently of structural alignment and orientation. Thus, this study aimed to evaluate the application of 3D Zernike descriptors for the structural clustering of antibodies and epitopes and to explore their use in antibody repurposing for the recognition of new targets. To this end, antibody binding sites previously associated with recognition of similar epitopes were analyzed at different structural levels, considering the CDRs, CDRH3, and complete paratopes. Surface similarity was subsequently quantified by calculating the Euclidean distance between their corresponding 3D Zernike feature vectors. Performance was benchmarked against SPACE2. Additionally, different distance thresholds were evaluated based on their ability to recover antibody pairs recognizing the same epitope. The paratope-based approach provided the best balance between the number of identified pairs and precision at a distance threshold of 2.7, whereas epitope clustering showed robust performance up to a distance of 3.0. At these thresholds, the 3D Zernike descriptors identified a greater number of functional pairs than SPACE2 while maintaining comparable precision and identifying complementary sets of antibody pairs.. BTaken together, these findings support the use of 3D Zernike descriptors for structural clustering of antibodies and epitopes and for guiding antibody repurposing G, a highly lethal zoonotic pathogen. Structural screening identified three antibodies with epitopes similar to the NiV target that also showed a consistent binding preference for the target epitope in molecular docking assays. Notably, one candidate, originally directed against a SARS-CoV-2 epitope, formed a stable complex with the NiV epitope, remaining within the 5 [A] RMSD threshold during heated molecular dynamics simulations and emerging as a potential cross-reactive candidate.These results support the use of this computational framework for biopharmaceutical discovery against emerging targets. Taken together, these findings support the use of 3D Zernike descriptors for structural clustering of antibodies and epitopes and for guiding antibody repurposing.

Carbapenems are the most potent {beta}-lactams, key antibiotics for healthcare-associated infections by Gram-negative bacteria and evade hydrolysis by most {beta}-lactamases, but are increasingly threatened by emergence of enzymes exhibiting hydrolytic activity towards them. Of the four recognised {beta}-lactamase subclasses, class A (active-site serine enzymes that hydrolyse {beta}-lactams via a covalent acylenzyme intermediate) is the most widely disseminated and, while the majority of such enzymes react with carbapenems to form long-lasting acylenzyme complexes, several possess carbapenem-hydrolyzing activity (carbapenemases). Here, we investigate the basis for these differences in a panel of class A {beta}-lactamases using molecular dynamics (MD) simulations of the respective acylenzyme complexes and tetrahedral intermediates (TI). The simulations reveal multiple features associated with catalytic activity across the spectrum of enzymes tested, including more extensive interactions of the carbapenem acylenzyme carbonyl and generally increased lifetimes of active site water molecules positioned for deacylation. Analysis of the dynamic trajectories shows carbapenemases to have reduced root mean-squared fluctuation (RMSF) differences between the acylenzyme and TI, that are not limited to the active site, indicating that the acylenzyme complex is pre-organised for reaction in carbapenemases but not in carbapenem-inhibited enzymes. Similarly, Principal Component Analysis (PCA) of acylenzyme and TI dynamics shows greater overlap between the two states in carbapenemases, providing further evidence for acylenzyme pre-organisation. Such simulations may represent an effective computational assay able to identify enzymes with carbapenemase activity at relatively modest computational cost.

Several potentially potent anticancer drugs have been identified by in vitro evaluation, such as Andrographolide. These compounds show strong anticancer activity in vitro, but struggle to reach effective concentrations in the bloodstream when taken orally because they dissolve poorly in water or break down rapidly in the body. Bioenhancers, which are compounds that have potential to improve drug stability in the body, offer an alternative solution to overcome this limitation. Naringin and Quercetin have been identified as candidate bioenhancers, and have been hypothesized to potentially slow rapid first pass metabolism of poorly bioavailable drugs. Our work focuses on testing Naringin and Quercetin because they are flavonoids with therapeutic potential, due to their anti-inflammatory and antioxidant properties. Data from the hepatic microsomal assays performed on Naringin and Quercetin suggest moderate to proficient periods of stability in the body, with Naringin having 91.86% remaining, while Quercetin had 74.84% remaining. When administered alongside Andrographolide, a drug known to rapidly degrade in the body, Naringin raised its metabolic stability from 38.93% to 80.77% and on the other hand, Quercetin raised Andrographolide metabolic stability from 38.93% to 86.70%. In addition, plasma protein binding assays show the percentage of compounds available at the target site where Naringin was observed to be 49.32% bound and Quercetin found to be 50.14% bound, implying 50.68% of Naringin, and 49.86% of Quercetin available at the target site, respectively. This preliminary study explores whether Quercetin and Naringin could act as bioenhancers by remaining stable and available in plasma and by slowing the metabolism of poorly bioavailable drugs such as Andrographolide.

Antisense oligonucleotides (ASO) are nucleotide polymers that hybridize to sense strands and have been successful in treating a variety of diseases. A wide range of strategies have been investigated to optimize and develop ASO for clinical studies. A key objective for this study was to provide an overview of the range of detailed data that get be obtained and provide an updated method review on how to design surface plasmon resonance (SPR) kinetic experiments for DNA oligonucleotide hybridization studies that can also be applied to other ASO including peptide nucleic acids (PNA). We describe many lessons learned from published literature and provide a state-of-the-art strategy and methods for generating not only kinetic but also thermodynamic characterizations of oligonucleotide hybridization. In this study we have performed an SPR kinetic and thermodynamic analysis for the hybridization of HIF1 antisense DNA strands to its immobilized Intron2-Exon3 splice site sense DNA strand to provide insight, in general, on the optimal length and insight into optimal design of DNA ASOs. We provide a process on how to design experiments to: 1.) obtain oligonucleotide-length dependent kinetics, 2.) analyze reactions to obtain association and dissociation rate kinetics (ka, kd), assess if hybridization follows a 2-state model and to obtain kinetic dissociation constants (Kd), 3.) perform temperature-dependent hybridization kinetics to obtain thermodynamic values ({Delta}H{degrees}, {Delta}S{degrees} and {Delta}G{degrees}) that can give insight into the molecular interactions driving hybridization, 4.) compare experimental thermodynamic values to values derived from nearest-neighbor prediction models to identify atypical reactions and importantly 5.) enable calculations to predict oligomer hybridization affinity at the physiological 37 {degrees}C temperature to asses if the design of the oligomer will have the required cellular activity for a therapeutic effect. The strategy and results presented throughout the paper are compared to previous SPR reports and suggestions made to optimize kinetic studies.

Kinema is a traditional fermented soybean food indigenous to the eastern Himalayan regions of Nepal and India. The fermentation process is primarily mediated by the bacterium Bacillus subtilis, which produces several bioactive compounds and enzymes with potential therapeutic applications. Considering the growing burden of cardiovascular diseases and the need for effective fibrinolytic agents for thrombolytic therapy, this study aimed to extract, partially purify, and evaluate the thrombolytic potential of kinemakinase derived from kinema prepared from white soybeans. Partial purification of the enzyme was achieved using ammonium sulfate precipitation. Thrombolytic activity was assessed in vitro using human blood clots, where three enzyme dilutions demonstrated clot lysis ranging from 66% to 68%, indicating considerable fibrinolytic potential. In silico analyses were also performed to investigate the structural and functional characteristics of the enzyme. The tertiary structure obtained from UniProt was modeled using the Robetta server and refined with GalaxyRefine. Docking with fibrin using ClusPro 2.0 and molecular dynamics simulations using iMODS confirmed favorable interaction and structural stability, while disulfide engineering enhanced protein stability. The findings suggest that kinema-derived kinemakinase may serve as a promising alternative thrombolytic agent, warranting further biochemical characterization and dosage optimization.

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

Green synthesis of zinc oxide nanoparticles (ZnO NPs) offers a sustainable strategy for developing multifunctional antimicrobial nanomaterials. In this study, ZnO NPs were synthesized using Azadirachta indica leaf extract and characterized by UV-vis spectroscopy, FTIR, XRD, SEM, and GC-MS. The nanoparticles exhibited a characteristic absorption peak at 352 nm, a direct band gap of 3.07 eV, and hexagonal wurtzite crystallinity with an average crystallite size of approximately 32 nm. The biosynthesized ZnO NPs showed concentration-dependent antibacterial activity against Erwinia carotovora, producing inhibition zones of up to 25.9 mm. Mechanistic studies revealed significant membrane damage, evidenced by 4.77-fold and 5.62-fold increases in extracellular protein and amino acid leakage, respectively, with marked alterations in bacterial protein profiles detected by SDS-PAGE. The nanoparticles also exhibited strong antioxidant activity, achieving 89.4% DPPH radical scavenging, and induced dose-dependent cytotoxicity in HepG2 cells with an estimated IC50 of 124.8 g/mL. These findings demonstrate that neem-mediated ZnO nanoparticles possess potent antibacterial activity through membrane disruption while exhibiting promising antioxidant properties, highlighting their potential as eco-friendly nanomaterials for the management of bacterial soft rot and other phytopathogenic diseases.

SH2 domains are phosphotyrosine-binding modules that play a critical role in cell signaling by mediating protein-protein interactions. While tyrosine phosphorylation has been shown to impact SH2 domain function in signaling, the specific effects of phosphorylation at different sites within the domain remain poorly understood. In this study, we selected two conserved regions of tyrosine phosphorylation within SH2 domains, near conserved binding interface residues, and developed approaches to evaluate the impact of those sites on ligand binding. Using a modified dot blot assay to screen phosphomimic mutations, we studied specific tyrosine residues within the PTPN11-N, LYN, and SYK-C SH2 domains, finding that the PTPN11 N-terminal site (Y63) modulates the specificity, reducing binding of physiologically relevant substrates. Our findings provide new insights into the regulatory mechanisms governing SH2 domain function and highlight the importance of site-specific phosphorylation in modulating protein-protein interactions in cell signaling pathways.

Complete loss of function of the voltage-gated sodium channel subtype Nav1.7, encoded by SCN9A, results in congenital insensitivity to pain. Here, we investigate a previously identified variant, M899I, in which methionine at position 899 is substituted by isoleucine. This variant was originally described in a Chinese patient with loss of pain. We confirmed membrane expression of the mutant channel in HEK cells using extracellular HA-tagging; however, no sodium currents were detectable from the variant in patch-clamp recordings. The M899I substitution is located within a tightly packed hydrophobic region of the pore module. Introducing the corresponding variant into Nav1.2 and Nav1.5 similarly abolished channel function, underscoring the high conservation and functional importance of this residue. To further investigate the underlying mechanism, we combined in-silico coarse-grained molecular dynamics simulations with in-vitro electrophysiological analysis. Our simulations predicted that the M899I substitution induces collapse of the outer pore, substantially reducing both pore radius and volume. Substitution with other hydrophobic residues was likewise predicted to alter pore geometry and, consequently, ion permeation to varying degrees. Whole-cell voltage-clamp recordings validated these predictions, with observed current densities closely correlating with the extent of pore collapse predicted in silico. Together, our findings establish pore collapse as a mechanism underlying disease-relevant loss-of-function variants in Nav1.7 and suggest that this principle may extend to other sodium channel subtypes. Moreover, our results demonstrate that in-silico molecular dynamics approaches can reliably predict structural and functional consequences of channel mutations, as confirmed by in-vitro electrophysiological data.