Pore-forming colicins are bacteriocins produced by Escherichia coli to kill competing bacterial strains by forming ion-permeable channels in the inner membrane of target cells, leading to membrane depolarization, ion leakage, and ultimately cell death. While the crystal structures of their soluble form and membrane-perforating activities have been intensively studied, the structural rearrangements enabling outer membrane translocation and inner membrane pore formation remain puzzling. Here, we present the crystal structure of the channel-forming domain of colicin Ib (ColIb) and identify interhelical salt bridge networks that stabilize its tertiary structure. Comparative analysis shows that electrostatic interactions between helices H3-H7 and H4-H6 are conserved in E1-type but not A-type colicins. Disrupting these electrostatic interactions-either through alanine substitutions or acidic pH-produced a less compact structure with increased membrane association. Salt bridge mutations enhanced the bactericidal activity of full-length ColIb by at least an order of magnitude, and notably, introducing these mutations into the isolated C-domain conferred CirA-dependent cytotoxicity in the absence of the T- and R-domains. Protonation of the C-domain at pH 4.5 further amplified its killing capacity. Our findings reveal that destabilization of interhelical contacts facilitates unfolding and membrane association, providing a structural mechanism for CirA-mediated translocation and amplified killing efficiency.

Introduction: Taiwan green propolis (TGP) is rich in prenylflavonoids and exhibits antioxidant, antibacterial, antiviral, and antitumour properties. It induces apoptosis in various cancer cells, making it a highly promising natural medicine. Although the health benefits and food applications of TGP are widely recognised, no study has explored its effects on Taiwan oral cancer cells (OECM1). This study investigated whether TGP induces apoptosis in OECM1 cells. Methods: High-performance liquid chromatography (HPLC), thin-layer chromatography, and liquid chromatography/mass spectrometry were used to identify the components in TGP and the fruit peel of Macaranga tanarius. The inhibitory activities of TGP dissolved in DMSO (TGPDMSO) and encapsulated in food-grade zein nanoparticles (TGPNP) against OECM1 cells were compared using MTT assays. The morphological changes, cell cycle analysis, and protein expression profiles of OECM1 cells after the TGP treatments were performed using microscopy, flow cytometry, and Western blot, respectively. Results: An MTT assay of TGPDMSO-treated OECM1 cells suggested an IC50 of 12.6 µg/mL, demonstrating that TGPDMSO exhibits significant cytotoxicity. Subsequent MTT assays revealed TGPNP’s cytotoxicity against OECM1 with an IC50 of 11.6 µg/mL. Flow cytometry revealed that TGPNP induced a cell arrest in S phase and DNA fragmentation. Western blotting analyses manifested an increase in Bax and cl-Casp9 and a decrease in Bcl2 and PARP. Conclusion: This study demonstrated that both TGPDMSO and TGPNP treatments induced apoptosis in OECM1 cells with a comparable IC50. Notably, utilising edible zein as a nanoparticle carrier for TGP mitigates the cytotoxicity risk associated with DMSO, providing a novel and safe approach for cancer treatment.

In untargeted metabolomics, high-resolution liquid chromatography–tandem mass spectrometry (HR-LC-MS/MS) is widely applied to achieve broad metabolite coverage. However, a single acquisition mode often presents trade-offs between coverage and quantification. To overcome this, we propose a dual-injection strategy combining full scan (FS) and data-dependent acquisition (DDA) modes in consecutive runs of the same sample. The resulting datasets are processed by an in-house developed algorithm that integrates feature lists from both FS and DDA, enhancing both the number of detected features and the quantification precision. This integrated approach, named Integrated Feature Set from DDA and FS (IFSDDA), improves peak picking robustness and captures features otherwise missed by conventional methods. A total of 32,830 metabolic features were detected using IFSDDA, markedly surpassing those detected by traditional approaches. Quantitative precision was ensured through QC-based support vector regression (SVR) normalization, with 94.2% of features achieving a relative standard deviation (RSD) below 20%. For metabolite annotation, 2845 features were structurally annotated, indicating superior annotation efficiency. Notably, the IFSDDA strategy facilitated the identification of six anti-MRSA metabolites from Pseudomonas aeruginosa, including 1-carboxamide (PCN), pyocyanin (PYO), 1-hydroxyphenazine (1-HP), PQS, and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). These metabolites were subsequently validated through multiple reaction monitoring (MRM) and biological activity assays. Overall, IFSDDA effectively addresses key limitations in untargeted metabolomics by enhancing data quality, reproducibility, and metabolite annotation. This strategy represents a significant advancement for comprehensive metabolic profiling and holds strong potential for biomarker discovery in complex biological systems.

Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain barriers to obtaining high-resolution structures persist, particularly during grid preparation when samples are in a thin liquid film. At this stage, extensive exposure to the air-water interface (AWI) can lead to subunit dissociation, denaturation, and preferred orientation of particles. Another obstacle to high-resolution cryoEM is molecular flexibility, which introduces heterogeneity in the dataset, weakening the signal during image processing. This study explores the effects of AWI interactions and molecular flexibility on the cryoEM density maps of KtrA, the soluble regulatory subunit of the potassium transporter KtrAB from Bacillus subtilis. From grids prepared using a standard blotting technique, we observed a lack of density in the C-lobe domains and preferred orientation. Modifications such as reducing AWI exposure through faster vitrification times (6 s vs ≤100 ms) notably improved C-lobe density. Moreover, the addition of cyclic di-AMP, which binds to the C-lobes, combined with a 100 ms plunge time, further enhanced C-lobe density and eliminated preferred orientation. These findings demonstrate that both AWI interactions and flexibility had to be addressed to obtain density for the C-lobe domains of KtrA. This study underscores the ongoing complexities in achieving high-resolution cryoEM for many samples.

Pathogenic Staphylococcus aureus endures bursts of host-derived reactive nitrogen species, yet the molecular defenses that enable this resilience have remained unclear. We now show that the previously enigmatic di-iron enzyme ScdA functions as a nitrite reductase, converting nitrite to nitric oxide (NO), and we elucidate the structural elements that support this activity. Using an integrative toolkit─X-ray crystallography, solution NMR, AlphaFold modeling, and pulsed EPR/DEER─we solved the full-length homodimeric structure of ScdA and identified a robust di-iron center that forms stable iron-nitrosyl intermediates. Targeted mutagenesis reveals that redox-active cysteines and dimerization state tune catalytic output, whereas steady-state kinetics confirm efficient nitrite-to-NO turnover. In vivo, ScdA overexpression in Escherichia coli suppresses growth under nitrite-rich conditions, highlighting the cytotoxic potency of the NO it generates. By coupling structure to function, our work clarifies S. aureus strategies for managing nitrosylative stress and points to ScdA as a potential vulnerability in antibiotic-resistant pathogens.

The HIRA complex, composed of HIRA, UBN1, and CABIN1 in humans, plays a central role in histone chaperone activity and chromatin regulation by depositing the H3.3 histone variant into nucleosomes. Proper subunit interactions are critical for complex stability and function. In this study, we examine the interaction between Hir2 and Hpc2, the yeast homologs of HIRA and UBN1, using biochemical and structural approaches. We show that the N-terminal to the Hpc2-related domain (NHRD) of Hpc2 binds to the WD40 domain of Hir2, consistent with the human HIRA-UBN1 interaction. The crystal structure of the Hir2_WD40-Hpc2_NHRD complex reveals a seven-bladed β-propeller fold in Hir2_WD40, with Hpc2_NHRD forming an antiparallel β sheet interface. Notably, a unique five-stranded blade in Hir2_WD40, stabilized by proline residue P228, is essential for Hpc2 binding. Mutational analysis confirms key interface residues, providing structural insights into the evolutionary conservation of the HIRA complex.

The study uses Sequential Window Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH)-MS in conjunction with secretome proteomics to identify key proteins that Pseudomonas aeruginosa secretes against methicillin-resistant Staphylococcus aureus (MRSA). Variations in the inhibition zones indicated differences in strain resistance. Multivariate statistical methods were applied to filter the proteomic results, revealing five potential protein biomarkers, including Peptidase M23. Gene ontology (GO) analysis and sequence alignment supported their antibacterial activity. Thus, SWATH-MS provides a comprehensive understanding of the secretome of P. aeruginosa in its action against MRSA, guiding future antibacterial research.

AbstractThe K+ uptake system KtrAB is essential for bacterial survival in low K+ environments. The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+ activates KtrAB and the Na+ binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na+, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na+ binding stabilizes the ATP-bound BsKtrAB complex and enhances its K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na+ in activating BsKtrAB is likely applicable to Na+-activated K+ channels in central nervous system.

Dengue virus (DENV) poses a significant global health challenge, with millions of cases each year. Developing effective antiviral drugs against DENV remains a major hurdle. Varenicline is a medication used to aid smoking cessation, with anti-inflammatory and antioxidant effects. In this study, varenicline was investigated for its antiviral potential against DENV. This study provides evidence of the antiviral activity of varenicline against DENV, regardless of the virus serotype or cell type used. Varenicline demonstrated dose-dependent effects in reducing viral protein expression, infectivity, and virus yield in Vero and A549 cells infected with DENV-1 and DENV-2, with EC50 values ranging from 0.44 to 1.66 μM. Time-of-addition and removal experiments demonstrated that varenicline had a stronger inhibitory effect on the post-entry stage of DENV-2 replication than on the entry stage, as well as the preinfection and virus attachment stages. Furthermore, cell-based trans-cleavage assays indicated that varenicline dose-dependently inhibited the proteolytic activity of DENV-2 NS2B-NS3 protease. Docking models revealed the formation of hydrogen bonds and van der Waals forces between varenicline and specific residues in the DENV-1 and DENV-2 NS2B-NS3 proteases. These results highlight the antiviral activity and potential mechanism of varenicline against DENV, offering valuable insights for further research and development in the treatment of DENV infection.

The coding region for the sortase A (SrtA) of Staphylococcus aureus was fused at the N-terminus of LfcinB. The SrtA-LfcinB fusion protein in E. coli C43(DE3) was expressed with the expected sizes of 21 kDa and 38 kDa by pET21b-SrtA-LfcinB and pET32-1SrtA-LfcinB constructs, respectively. Increased levels of the TrxA-His-SrtA-SrtA-LfcinB fusion protein were detected by the pET32-3SrtA-LfcinB construct having three expression cassettes. LfcinB is released from the expressed SrtA-LfcinB protein by SrtA self-cleavage which is induced in the presence of Ca2+. The antibacterial activity was detected after SrtA-mediated cleavage of LfcinB. Furthermore, to reduce the antimicrobial peptide toxicity to the E. coli host, the human interferon-γ (hIFN-γ) sequences were mutated into a negatively charged mIFc2 protein (7 kDa), which was co-expressed with LfcinB in an insoluble form. The yield of LfcinB was elevated while changing the gene order of LfcinB and mIFc2 (pET21b-fLfcinB-bmIFc2). Furthermore, increased levels of LfcinB were detected using the pET21b-(fLfcinB-bmIFc2)2 construct. To increase the dissolution rate of inclusion bodies, inclusion bodies treated with different temperatures and pH and resuspended in different volumes of 50 mM Tris-HCl were assayed. Our results reveal that heat-treated LfcinB/mIFc2 inclusion bodies at 90 °C, pH 10, and 16X resuspended volumes have the best resolubilization rate. This work suggests that the mIFc2 co-expression system shows higher efficiency for LfcinB production than the SrtA fusion system. The expressed LfcinB from the mIFc2 co-expression system exhibits excellent broad-spectrum antibacterial activities against thirteen Gram-negative and ten Gram-positive bacteria species with a range of minimum inhibitory concentrations (MIC) between 37–150 ug/mL.

Background: The global rise in nosocomial infections associated with gram -negative bacteria and the spread of multi-drug resistant Acinetobacter baumannii (MDR-AB) pose public health concerns. This study investigates the inhibitory effects and possible inhib-itory mechanism of Pseudomonas aeruginosa (PA) on selected clinical strains of A. baumannii (AB) isolated from Taiwanese patients. Methods: Four and eight clinical strains of AB and PA, respectively, were randomly selected from the bacterial collection of Feng-Yuan Hospital, Taiwan. Antimicrobial-susceptibility was performed on the AB strains. Inhibition potential of the PA strains against AB was assessed by measuring the inhibition zones. In vitro analysis using phenazine-1-carboxamide (PCN) was conducted to assess the possible inhibitory mechanism of PA, which was later confirmed in the clinical isolates by liquid chromatography-mass spectrometry. Results: All the clinical AB strains showed resistance to the eleven antibiotics and were classified as MDR-AB. The nine PA strains exert either a high (PA3596, PA3681, PA3772, and ATCC27853) or a low (PA3613, PA3625, PA3712, PA3715, and PA3744) degree of inhibition against AB strains. 0.25 mg/ml PCN had a clearer inhibition zone than 0.05 mg/ml PCN, suggesting a dose-dependent inhibition of PCN on the AB strains. The four PA strains that demonstrated a high degree of inhibition had a relatively high amount of PCN. Conclusion: Selected strains of PA exert inhibitory actions on MDR-AB with PCN being a possible inhibitory agent. This finding raises the possibility of developing effective therapeutic antibiotics and disinfectant from specific components of PA for the treatment and control of Acinetobacter-associated infections in hospital settings. Copyright 2020, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Biocatalysis refers to the utilization of enzymes, either in purified form, or existed as part of crude cell lysate or intact cells, to catalyze single- or multi-step chemical reactions, converting synthetic molecules or natural metabolites into high-value products [...]

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na+ as the driving force. The crystal structures of two bacterial homologues ASBTNM and ASBTYf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na+-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na+ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBTNM under different Na+ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBTNM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na+ ions shift the conformational equilibrium of ASBTNM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application.

Avian reoviruses (ARVs) are important pathogens that cause considerable economic losses in poultry farming. To date, host factors that control stabilization of ARV proteins remain largely unknown. In this work we determined that the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC) is essential for avian reovirus (ARV) replication by stabilizing outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV. TriC serves as a chaperone of viral proteins and prevent their degradation via the ubiquitin-proteasome pathway. Furthermore, reciprocal co-immunoprecipitation assays confirmed the association of viral proteins (σA, σC, and σNS) with TRiC. Immunofluorescence staining indicated that the TRiC chaperonins (CCT2 and CCT5) are colocalized with viral proteins σC, σA, and σNS of ARV. In this study, inhibition of TRiC chaperonins (CCT2 and CCT5) by the inhibitor HSF1A or shRNAs significantly reduced expression levels of the σC, σA, and σNS proteins of ARV as well as virus yield, suggesting that the TRiC complex functions in stabilization of viral proteins and virus replication. This study provides novel insights into TRiC chaperonin governing virus replication via stabilization of outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV.

Lignocellulosic biomass conversion technology seeks to convert agricultural waste to sugars through the use of various cellulases and hemicellulases. In practice, the application of free enzymes might increase the cost of the process due to difficulties with recovery of the enzymes and products. Immobilization might be an effective approach for recovering the hydrolysis products and improving the stability and reusability of the enzymes. In this study, we used a recombinant genetic engineering approach to construct a scaffold protein gene (CipA) and a xylanase gene (XynC) fused to a dockerin gene (DocT). After expressing CipA and XynC-DocT (XynCt) genes using E. coli hosts, the crude extracts were collected. An immobilized metal ion affinity membrane/Co2+ ion (IMAM-Co2+) system was prepared to adsorb CipA in its crude extract, thereby allowing simultaneous purification and immobilization of CipA protein. A similar approach was applied for the adsorption of XynCt protein, exploiting the interaction between the cohesin units in IMAM-Co2+-CipA and the dockerin unit in XynCt. The activity of the xylanase unit was enhanced in the presence of Co2+ for both the free XynCt enzymes and the immobilized CipA-XynCt. The heat resistance and stability over a wide range of values of pH of the immobilized CipA-XynCt were superior to those of the free XynCt. Furthermore, the immobilized CipA-XynCt retained approximately 80% of its initial activity after seven reaction cycles. The values of Km and νmax of IMAM-Co2+-CipA-XynCt (1.513 mg/mL and 3.831 U/mg, respectively) were the best among those of the other tested forms of XynCt.

In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.

The topology of helix-bundle membrane proteins provides low-resolution structural information with regard to the number and orientation of membrane-spanning helices, as well as the sidedness of intra/ extra-cellular domains. In the past decades, several strategies have been developed to experimentally determine the topology of membrane proteins. However, generally, these methods are labour-intensive, time-consuming and difficult to implement for quantitative analysis. Here, we report a novel approach, site-directed alkylation detected by in-gel fluorescence (SDAF), which monitors the fluorescent band shift caused by alkylation of the EGFP-fused target membrane protein bearing one single introduced cysteine. In-gel fluorescence provides a unique readout of target membrane proteins with EGFP fusion from non-purified samples, revealing a distinct 5 kDa shift on SDS-PAGE gel due to conjugation with mPEG-MAL-5K. Using the structurally characterised bile acid transporter ASBT(NM) as an example, we demonstrate that SDAF generates a topology map consistent with the crystal structure. The efficiency of mPEG-MAL-5K modification at each introduced cysteine can easily be quantified and analysed, providing a useful tool for probing the solvent accessibility at a specific position of the target membrane protein.

The Staphylococcus epidermidis lipase (SeLip, GehC) can be used in flavour-compound production via esterification in aqueous solution. This study reports the crystallization and crystallographic analysis of recombinant GehC (rGehC; Lys303-Lys688) with a molecular weight of 43 kDa. rGehC was crystallized at 293 K using PEG 10 000 as a precipitant, and a 99.9% complete native data set was collected from a cooled crystal at 77 K to a resolution of 1.9 Å with an overall Rmerge value of 7.3%. The crystals were orthorhombic and belonged to space group P212121, with unit-cell parameters a = 42.07, b = 59.31, c = 171.30 Å, α = β = γ = 90°. Solvent-content calculations suggest that there is likely to be one lipase subunit in the asymmetric unit.

Repetitive DNA sequences are ubiquitous in life, and changes in the number of repeats often have various physiological and pathological implications. DNA repeats are capable of interchanging between different noncanonical and canonical conformations in a dynamic fashion, causing configurational slippage that often leads to repeat expansion associated with neurological diseases. In this report, we used single-molecule spectroscopy together with biophysical analyses to demonstrate the parity-dependent hairpin structural polymorphism of TGGAA repeat DNA. We found that the DNA adopted two configurations depending on the repeat number parity (even or odd). Transitions between these two configurations were also observed for longer repeats. In addition, the ability to modulate this transition was found to be enhanced by divalent ions. Based on the atomic structure, we propose a local seeding model where the kinked GGA motifs in the stem region of TGGAA repeat DNA act as hot spots to facilitate the transition between the two configurations, which may give rise to disease-associated repeat expansion.