Structure-function Relationships Research Articles

RNA sample optimization for cryo-EM analysis.

RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine these 3D structures due to their conformational heterogeneity and intrinsic dynamics. Cryogenic electron microscopy (cryo-EM) has recently played an emerging role in resolving dynamic conformational changes and understanding structure-function relationships of RNAs including ribozymes, riboswitches and bacterial and viral noncoding RNAs. A variety of methods and pipelines have been developed to facilitate cryo-EM structure determination of challenging RNA targets with small molecular weights at subnanometer to near-atomic resolutions. While a wide range of conditions have been used to prepare RNAs for cryo-EM analysis, correlations between the variables in these conditions and cryo-EM visualizations and reconstructions remain underexplored, which continue to hinder optimizations of RNA samples for high-resolution cryo-EM structure determination. Here we present a protocol that describes rigorous screenings and iterative optimizations of RNA preparation conditions that facilitate cryo-EM structure determination, supplemented by cryo-EM data processing pipelines that resolve RNA dynamics and conformational changes and RNA modeling algorithms that generate atomic coordinates based on moderate- to high-resolution cryo-EM density maps. The current protocol is designed for users with basic skills and experience in RNA biochemistry, cryo-EM and RNA modeling. The expected time to carry out this protocol may range from 3 days to more than 3 weeks, depending on the many variables described in the protocol. For particularly challenging RNA targets, this protocol could also serve as a starting point for further optimizations.

RASP v2.0: an updated atlas for RNA structure probing data.

RNA molecules function in numerous biological processes by folding into intricate structures. Here we present RASP v2.0, an updated database for RNA structure probing data featuring a substantially expanded collection of datasets along with enhanced online structural analysis functionalities. Compared to the previous version, RASP v2.0 includes the following improvements: (i) the number of RNA structure datasets has increased from 156 to 438, comprising 216 transcriptome-wide RNA structure datasets, 141 target-specific RNA structure datasets, and 81 RNA-RNA interaction datasets, thereby broadening species coverage from 18 to 24, (ii) a deep learning-based model has been implemented to impute missing structural signals for 59 transcriptome-wide RNA structure datasets with low structure score coverage, significantly enhancing data quality, particularly for low-abundance RNAs, (iii) three new online analysis modules have been deployed to assist RNA structure studies, including missing structure score imputation, RNA secondary and tertiary structure prediction, and RNA binding protein (RBP) binding prediction. By providing a resource of much more comprehensive RNA structure data, RASP v2.0 is poised to facilitate the exploration of RNA structure-function relationships across diverse biological processes. RASP v2.0 is freely accessible at http://rasp2.zhanglab.net/.

Highly Tunable, Nanomaterial-Functionalized Structural Templating of Intracellular Protein Structures Within Biological Species.

Inside living organisms, proteins are self-assembled into diverse 3D structures optimized for specific functions. This structure-function relationship can be exploited to synthesize functional materials through biotemplating and depositing functional materials onto protein structures. However, conventional biotemplating faces limitations due to the predominantly intracellular existence of proteins and associated challenges in achieving tunability while preserving functionality. In this study, Conversion to Advanced Materials via labeled Biostructures (CamBio), an integrated biotemplating platform that involves labeling target protein structures with antibodies followed by the growth of functional materials, ensuring outstanding nanostructure tunability is proposed. Protein-derived plasmonic nanostructures created by CamBio can serve as precise quantitative tools for assessing target species is demonstrated. The assessment is achieved through highly tunable and efficient surface-enhanced Raman spectroscopy (SERS). CamBio enables the formation of dense nanogap hot spots among metal nanoparticles, templated by diverse fibrous proteins comprising densely repeated monomers. Furthermore, iterative antibody labeling strategies to adjust the antibody density surrounding targets, amplifying the number of nanogaps and consequently improving SERS performance are employed. Finally, cell-patterned substrates and whole meat sections as SERS substrates, confirming their easily accessible, cost-effective, scalable preparation capabilities and dimensional tunability are incorporated.

Induction of ectosome formation by binding of phospholipases D from Loxosceles venoms to endothelial cell surface: Mechanism of interaction.

Members of the phospholipase D (PLD) superfamily found in Loxosceles spider venoms are potent toxins with inflammatory and necrotizing activities. They degrade phospholipids in cell membranes, generating bioactive molecules that activate skin cells. These skin cells, in turn, activate leukocytes involved in dermonecrosis, characterized by aseptic coagulative necrosis. Although the literature has advanced in understanding the structure-function relationship, the cell biology resulting from the interactions of these molecules with cells remains poorly understood. In this study, we show that different cells exposed to recombinant PLDs bind these molecules to their plasma membrane, leading to the subsequent organization of extracellular microvesicles/ectosomes. The binding occurs as quickly as five minutes or less after exposure, increases over time, and eventually, the PLDs are expelled from the cell surface without generating cytotoxicity. PLDs are not endocytosed, nor do they spatially colocalize with acidic organelles in the intracellular environment. At least two regions of PLDs - the domain involved in magnesium ion coordination and the choline binding site - appear to play a role in cell surface binding and ectosome organization. However, the amino acids involved in catalysis do not participate in these events. The binding of these PLDs to the cell membrane, independent of catalytic activity, is sufficient to trigger intracellular signaling and enhance the expression of the pro-inflammatory IL-8 gene. These results are supported by the observation that isoforms of PLDs lacking catalytic activity induce an inflammatory response in vivo when injected into the skin of rabbits, without causing dermonecrosis. Our data indicate that these PLDs bind to the surface of target cells, promoting the organization of extracellular vesicles/ectosomes. Subsequently, these events activate pro-inflammatory genes and induce an inflammatory response in vivo. The binding to cells is not dependent on amino acids involved in catalysis but rather on amino acids involved in magnesium coordination. The binding of PLDs to the cell surface, formation of ectosomes, and activation of cells appear to initiate signals involved in inflammatory responses that can lead to dermonecrosis in accidents. This correlation is supported by experimental observations indicating that the events of toxin binding to cells, formation of microvesicles, and inflammatory responses observed both in vitro and in vivo are interconnected.

A Workflow for Creating Gastric Computational Models from SPARC Scaffolds

In-silico studies are an ideal medium to model and improve our understanding of the mechanisms underlying gastric motility in health and disease. In this study, a workflow to create computational models of the stomach was developed using SPARC scaffolds. Three anatomically based finite element method (FEM) models of the rat stomach incorporating experimental measurements of muscle layer thickness and fiber orientations across the stomach were developed: (i) 2D (surface) FEM model with no thickness, (ii) 3D (volume) FEM model with a fixed thickness across the longitudinal and circular muscle layers, and (iii) 3D (volume) FEM model with varying thickness across the longitudinal and circular muscle layers. The three FEM models were subsequently used in whole-organ slow wave simulations and the impact of anatomical details on the simulation outcomes was investigated. The 3D FEM model with varying thickness was the most computationally expensive, while the 2D FEM model provided the fastest solution (a 200 s simulation took 8 min vs. 38 h to solve). The spatiotemporal profiles of the slow wave activation and propagation in the three FEM models were in good agreement. The largest temporal difference of 1 s in cellular activation was observed between the 2D FEM model and the varying thickness 3D FEM model in the most distal-stomach regions. These FEM models and developed workflow will be used in in-silico studies to improve our understanding of the structure-function relationship in the stomach and identify the optimal parameters of electrical therapies, an alternative treatment for the motility disorders in the stomach. In addition, the developed workflow can be readily used to generate computational models of other organs using SPARC scaffolds.

Comparative Characterization of Three Homologous Glutathione Transferases from the Weed Lolium perenne

The comparative analysis of homologous enzymes is a valuable approach for elucidating enzymes’ structure–function relationships. Glutathione transferases (GSTs, EC. 2.5.1.18) are crucial enzymes in maintaining the homeostatic stability of plant cells by performing various metabolic, regulatory, and detoxifying functions. They are promiscuous enzymes that catalyze a broad range of reactions that involve the nucleophilic attack of the activated thiolate of glutathione (GSH) to electrophilic compounds. In the present work, three highly homologous (96–98%) GSTs from ryegrass Lolium perenne (LpGSTs) were identified by in silico homology searches and their full-length cDNAs were isolated, cloned, and expressed in E. coli cells. The recombinant enzymes were purified by affinity chromatography and their substrate specificity and kinetic parameters were determined. LpGSTs belong to the tau class of the GST superfamily, and despite their high sequence homology, their substrate specificity displays remarkable differences. High catalytic activity was determined towards hydroxyperoxides and alkenals, suggesting a detoxification role towards oxidative stress metabolites. The prediction of the structure of the most active LpGST by molecular modeling allowed the identification of a non-conserved residue (Phe215) with key structural and functional roles. Site-saturation mutagenesis at position 215 and the characterization of eight mutant enzymes revealed that this site plays pleiotropic roles, affecting the affinity of the enzyme for the substrates, catalytic constant, and structural stability. The results of the work have improved our understanding of the GST family in L. perenne, a significant threat to agriculture, sustainable food production, and safety worldwide.

HOST-GUEST INTERACTIONS FACILITATED BY CHALCOGEN BONDING WITHIN SELENADIAZOLE FUNCTIONALISED PORPHYRIN NANOTUBES.

The structural rigidity of tetrakis(4-pyridyl)porphyrin (TPyP) has been utilised to prepare a robust novel porous coordination polymer of composition Cd2(TPyP)(sez)2 (TPyP = 5,10,15,20-tetra(4-pyridyl)porphyrin, sez = 1,2,5-benzoselenadiazole-5-carboxylate). The coordination polymer may be described as a hexagonal porphyrin nanotube (PNT) and has the potential to bind guest molecules through chalcogen bonding. Single crystal X-ray diffraction (SCXRD) data indicate an internal pore diameter ~9 Å which represents ~35% of the crystal volume. Immersion of the PNTs in solvents such as DMSO and CS2 result in the incorporation of these molecules within the nanotubes with chalcogen bonding between host and guest. The crystallographic guest-inclusion investigations are complemented by solid-state 77Se, 13C, 113Cd and 2H NMR studies which provide insights into dynamic behaviour. The porosity of the crystals was further explored using gas adsorption experiments, indicating the reversible uptake of CO2, CH4, H2 and N2. Structure-function relationships are clearly established from complementary crystallographic, NMR and adsorption investigations.

Hydrophobic and Elastic Structural Triboelectric Materials Enabled by Template Method toward Real-Time Material Recognition.

Since each material has a unique ability to lose or obtain electrons, specific triboelectric signals are produced when triboelectric materials are in contact with different objects. Triboelectric nanogenerator (TENG) devices show great potential for use as tactile sensors; nevertheless, analyzing the structure-function relationship of functionalized triboelectric sensing interfaces under environmental conditions and improving the sensing stability and accuracy through the design of hydrophobic structure on the triboelectric material surface remain major challenges in the development of intelligent sensing networks. Compared with the traditional rigid micronanostructure, the elastic micronanostructure strategy is applied to achieve both hydrophobicity and stability of triboelectric materials based on the template method in this work. The corresponding surface roughness and contact angle are 89.9 nm and 117.9°, respectively. As expected, the output voltage and charge density are enhanced by almost 65.8 and 33.4%, respectively, with the establishment of an elastic micronanostructure on the triboelectric material surface. More importantly, the triboelectric signal waveforms also present acceptable durability for subsequent recognition after immersion in water or ethanol for 12 days and metal impact for 12 000 cycles. Hence, combined with deep machine learning and triboelectric effect, a material perception system integrated with a moisture-resistant TENG-based sensor after fatigue testing, data processing, and display modules is also developed for real-time monitoring with approximately 100% (mask), 76% (plank), 93% (plastic), and 89% (rubber) identification accuracies in the natural environment. Finally, the proposed hydrophobic and elastic triboelectric materials show broad potential for application in the field of human-computer interaction.

TMEM16 and OSCA/TMEM63 proteins share a conserved potential to permeate ions and phospholipids

The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, are thought to function solely as ion channels. However, most TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure–function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here, we show that single mutations along the transmembrane helix (TM) 4/6 interface allow non-scrambling TCS members to permeate phospholipids. In particular, this study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.

High-throughput synthesis and optimization of ionizable lipids through A3 coupling for efficient mRNA delivery

BackgroundThe efficacy of mRNA-based vaccines and therapies relies on lipid nanoparticles (LNPs) as carriers to deliver mRNA into cells. The chemical structure of ionizable lipids (ILs) within LNPs is crucial in determining their delivery efficiency.ResultsIn this study, we synthesized 623 alkyne-bearing ionizable lipids using the A3 coupling reaction and assessed their effectiveness in mRNA delivery. ILs with specific structural features—18-carbon alkyl chains, a cis-double bond, and ethanolamine head groups—demonstrated superior mRNA delivery capabilities. Variations in saturation, double bond placement, and chain length correlated with decreased efficacy. Alkynes positioned adjacent to nitrogen atoms in ILs reduced the acid dissociation constant (pKa) of LNPs, thereby hindering mRNA delivery efficiency. Conversion of alkynes to alkanes significantly enhanced mRNA delivery of ILs both in vitro and in vivo. Moreover, combining optimized ILs with cKK-E12 yields synergistic LNPs that showed markedly augmented mRNA expression levels in vivo.ConclusionsOverall, our study provides insights into the structure–function relationships of ILs, providing a foundation for the rational design of ILs to enhance the efficacy of LNPs in mRNA delivery.Graphical

TMEM16 and OSCA/TMEM63 proteins share a conserved potential to permeate ions and phospholipids.

The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, are thought to function solely as ion channels. However, most TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure-function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here, we show that single mutations along the transmembrane helix (TM) 4/6 interface allow non-scrambling TCS members to permeate phospholipids. In particular, this study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.

A historical perspective on the insect central complex: Anatomy, development, and function

The central complex (CX), a remarkable brain region at the core of insect behaviors, has been the subject of extensive research for decades. In this review, we offer a comprehensive historical perspective on the anatomy, development, and function of the CX. The CX consists of discrete and highly structured neuropils found at the center of the brain, which are conserved across insects and arthropods. The developmental processes that shape the CX are themselves conserved across insects. In early research, the CX had been shown to receive visual information and control motor function. Using increasingly advanced methods throughout the years, it has become clear that the CX is involved in high-level behavioral control, such as in vector navigation and goal directed orientation. Its numerical simplicity presents a rare opportunity to study structure-function relationships in small brains, gain insights into evolutionary neurobiology, and develop novel neuromorphic technologies inspired by insect brains. Since the CX is a highly conserved brain region involved in the control of spatial tasks, it is uniquely suited to gain a detailed understanding of the computations required for these processes at the level of neural circuits.

Thermally Bisignate Anion Responsive Supramolecular Gel and In Situ Generation of a Conductive Hybrid-Gel Nanocomposite.

Understanding the structure-function relationship is a significant challenge in designing supramolecular soft materials such as supramolecular gels. To address this challenge, we report on two urea-based dipodal ligands, PY-NAP and PY-CF3, with different terminal substituents influencing their gelation properties. The terminal substituents play a crucial role in the gelation abilities. The gel formed from PY-NAP exhibited notably high thermal stability and displayed a unique "thermally bisignate" behavior. Both ligands contain urea and amide units, allowing them to encapsulate the SO42- anion in their pincer cavities in the solid state. The solid-state anion recognition principle is used to construct a selective anion-responsive supramolecular gel. Additionally, the gel was used to sequester precious metal salts from aqueous solutions, achieving an uptake efficiency of over 90%, followed by in situ reduction to form nanoparticles. This concept was then applied to create a conductive supramolecular hybrid gel nanocomposite with significantly high conductivity, holding significant implications for industrial and environmental applications.

Synergistic promotion mechanism and structure-function relationship of nonmetallic atoms doped carbon nanodots driving Tagetes patula L. to remediate cadmium-contaminated soils

Phytoremediation is an economical and effective strategy to remove cadmium (Cd) from polluted environments. To improve its efficiency, nanotechnology has been proposed to collaborate with hyperaccumulators in the remediation of Cd-polluted soils. However, the intricate structure-function relationship and the underlying regulatory mechanisms by which nanomaterials regulate Cd migration and conversion within the soil-plant system remained unrevealed. In this study, functional carbon nanodots (FCNs) were modified by doping with nitrogen and (or) sulfur elements. The synthesized nonmetallic atoms-doped FCNs were utilized to investigate their structure-function relationship and the regulatory mechanisms underlying their role in the phytoremediation of Cd-polluted soils by Tagetes patula L. FCNs-based nanomaterials can regulate the migration and bioaccumulation of Cd in the soil-plant system, which exhibits an obvious structural dependency. Specifically, the synergistic application of sulfur doped FCNs and Tagetes patula L. had the highest Cd removal efficiency of 53.2%, which was 20.1% higher than Tagetes patula L. alone. The uptake and migration of Cd in the soil-plant system are regulated by FCNs-based nanomaterials through both direct and indirect mechanisms, involving interfacial reactions, plant physiology regulation and environmental influence. This study not only sheds light on the fate of FCNs-based nanomaterials and Cd in the soil-plant system, but also provides innovative nanotools for reinforcing phytoremediation efficiency in contaminated soils.

Impact of missense mutations on the structure–function relationship of human succinyl-CoA synthetase using in silico analysis

The encephalomyopathic mtDNA depletion syndrome with methylmalonic aciduria is associated with succinyl-CoA synthetase (SCS) deficiency caused by pathogenic variants in genes encoding its two subunits. SCS is a mitochondrial enzyme involved in several metabolic pathways and acts as a heterodimer composed of α and β subunits encoded by SUCLG1 and SUCLA2 genes, respectively. The purpose of this study was to analyze the effects of the most pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) by applying, using different prediction tools, a filtering strategy, on the 343 and 365 nsSNPs found in SUCLG1 and SUCLA2 genes, respectively, retrieved from the databases, then to evaluate their structural and functional effects using homology modeling and molecular docking. Results showed that most deleterious mutations selected for structural analysis were located in loop regions critical for protein stability and function, especially, variants altering glycine and proline residues in these regions supporting their importance. We also showed that variants leading to hydrophobic and hydrophilic residues can destabilize the folding and binding of the protein. Molecular docking has also been used to identify the most important regions of ligand binding site (CoA binding site, ADP-Mg2+ binding site and phosphate ion binding site) and between the two subunits themselves, which mainly involving the ligase CoA domain. Our structural analysis, performed on selected nsSNP, are in accordance with experimental studies reported in the literature and predicted that they would responsible to either nonfunctional protein, subunit instability resulting in reduced amounts of misassembled protein, or in a protein unable to phosphorylate ADP.

Highly dispersed single-atom Fe and Fe3O4 clusters anchored hierarchical-pore carbon as efficient persulfate activation catalyst

The selective removal of Fe particles from carbonized Fe-MOFs presents an intriguing strategy for developing efficient peroxydisulfate (PDS) activators. It is commonly accepted that their outstanding performance is largely attributed to the high specific surface area/pore volume. However, our investigation into the structure–function relationship of these materials has revealed new insights. In this study, a highly dispersed single-atom Fe and Fe3O4 clusters anchored hierarchical-pore carbon (CM88-H) was obtained via post-acid treatment of carbonized MIL-88B (CM88). Various characterizations were comparatively conducted to highlight the beneficial effects of iron dissolution. The CM88-H/PDS system achieved a notable BPA degradation efficiency, with a reaction rate of 0.323 min−1, which is 3.94 times higher than that of the CM88/PDS system. DFT calculations indicate that the Fe3O4 clusters optimize the charge distribution around single-atom Fe, promoting favorable PDS adsorption and facilitating a higher oxidation state of CM88-H. As a result, BPA can be completely degraded within 10 min with 0.1 g/L CM88-H and 0.05 g/L PDS via both radical and non-radical pathways. The CM88-H/PDS/BPA system performs effectively under various conditions, with a general reduction in toxicity. Furthermore, the CM88-H/PDS system shows broad oxidation activity towards BPA substitutes and pollutants with high ionization potential (IP). Notably, the CM88-H sponge-based fixed-bed catalytic reactor demonstrated satisfactory BPA degradation, highlighting the promising application of CM88-H in water purification. This study provides a sustainable solution for water treatment and accelerates the practical application of powdered nanocatalysts to improve water quality.

Structure-function relationship of PE11 esterase of Mycobacterium tuberculosis with respect to its role in virulence

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest (∼10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both α-helix and β-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a Gx3Sx4G motif homologous to canonical ‘GxSxG’ motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

Microanatomy sectioning and phytochemicals overview in selected Oleaceae species

This study provides in-depth observations into the microanatomical structure of leaves and stem of eight selected Oleaceae species revealing comprehensive tissue organization using microtome sectioning and peeling leaf epidermis techniques. The phytochemicals profile overview of selected olive species extracted from leaf and stem of Oleaceae species contributes to identifying bioactive compounds with potential therapeutic and medicinal applications. An overview of histochemistry of bioactive metabolites revealed in Oleacee species shows the presence of phenolic compounds (oleuropein and hydroxytyrosol), localized in the leaves and stems, contributing to their antioxidant properties. The present study represent foliar anatomical traits; epidermal cells (ECs) polygonal shape and straight anticlinal walls, paracytic stomata and simple unicellular trichomes. The maximum ECs length (30 μm) was noted in Olea europaea and minimum (20 μm) in Jasminum fluminense was measured. Oleaceae species microtome stem sectioning examined revealed cortex type (parenchyma and collenchyma), vascular bundles (VBs) arrangements (radial, scattered and bundled), xylem vessels (scalariform, annular, spiral and reticulate), phloem fiber arrangements (banded and scattered), and uniseriate, biseriat and multiseriate medullary rays. Stem transverse section with maximum length (1300 μm) was observed in Olea europaea and the lowest (1100 μm) in Jasminum grandiflorum L. subsp. floribundum. This taxonomic histological method links the relationship between anatomy and chemistry, lead to identification of bioactive chemical compounds diversity among Oleaceae species. This anatomical methods used to enhance and consider the plant micromorphological structure-function relationships adapted to diverse environmental conditions.

Strategic alteration of arabinoxylan feruloylation enables selective shaping of the human gut microbiota

Arabinoxylans (AXs) are promising prebiotic candidates abundant in cereals. While AX feruloylation impacts properties, its effects on long chains mimicking native structures are unclear. This study revealed a dose-dependent impact of long chain AX feruloylation on gut microbial composition and function. In vitro fecal fermentation with varying AX feruloylation showed increased Bacteroidetes and propionate along with decreased Firmicutes and butyrate. Distinct Bacteroides populations were enriched under different feruloylation levels, suggesting specialized adaptation. Community dynamics and co-occurrence networks highlighted intricate taxon-specific responses, underscoring the unique microbial profiles shaped by individual variations. This study lays the foundation for elucidating the metabolic pathways and enzymatic machineries enabling utilization of feruloylated AXs based on the dose-dependent enrichment of specific taxa. This knowledge can inform rational design of customized prebiotics through precision nutrition. This work provides novel insights into tailoring cereal biomass fermentation and microbiome structure-function relationships. It establishes a platform for developing optimized feruloylated prebiotics to deliberately modulate gut ecology and address intestinal dysbiosis.

Two pairs of back-to-back α-helices of Kingella kingae RtxA toxin are crucial for the formation of a membrane pore.

The RtxA cytotoxin, a member of the RTX (Repeats in ToXin) family of pore-forming toxins, is the primary virulence factor of the paediatric facultative pathogen Kingella kingae. Although structure-function studies of RTX toxins have defined their characteristic domains and features, the exact membrane topology of RTX toxins remains unknown. Here, we used labelling of cell-bound RtxA with a membrane-impermeable, lysine-reactive reagent and subsequent detection of the labelled lysine residues by mass spectrometry, which revealed that most of the membrane-bound toxin is localised extracellularly. A trypsin protection assay with cell-bound RtxA demonstrated that five of seven transmembrane α-helices, predicted by various algorithms within the N-terminal half of the molecule, are irreversibly embedded in the membrane. Structure-function analysis showed that these α-helices, four of which are arranged as two pairs of back-to-back helices, are essential for the formation of an ion-conducting membrane pore. In contrast, the C-terminal half of RtxA is required for the interaction with the cell surface and for the irreversible insertion of the toxin into the membrane via acyl chains covalently linked to the molecule. These findings advance our understanding of the structure-function relationships of RtxA and enable us to propose a membrane topology model of the toxin.