International Journal of Biological Macromolecules

1.Iota-carrageenan is a natural polymer with moisturizing, mucoadhesive and shear-thinning properties. In this study, we aimed to evaluate the protective effects of iota-carrageenan on ocular surface against dehydration, to demonstrate its suitability for the use in lubricant eye drops. We utilized a human epithelial corneal cell culture model to test if pre-incubation with iota-carrageenan solution could protect cells from desiccation-induced cell death, to compare its effect with other natural polymers commonly used in artificial tear products, and to determine the optimal iota-carrageenan concentration. An ex vivo porcine eye model was established to confirm the protective effect of iota-carrageenan against dehydration on ocular tissue. Pre-incubation with 1.2 mg/ml iota-carrageenan increased the survival half-life of human corneal epithelial cells upon dehydration by three-fold; the effect was in the same range as observed for large molecular weight hyaluronic acid, and superior to all other tested natural polymers. The highest tested concentration of iota-carrageenan, 1.6 mg/ml, extended the cellular survival half-life by eight-fold while maintaining healthy cellular morphology. Repeated ex vivo instillation of an iota-carrageenan-based ophthalmic formulation into porcine eyes significantly protected the ocular surface from desiccation-induced corneal damage, as shown by corneal fluorescein staining These data suggest that Iota-carrageenan effectively moisturizes and protects the ocular surface, supporting its potential as a promising novel ingredient for eye drops in the management of dry eye disease.

Hyaluronic acid (HA) is a biologically versatile polysaccharide synthesized by vertebrates and several microbial pathogens. To date, Cryptococcus neoformans CPS1p is the only reported hyaluronic acid synthase (HAS) in fungi, which is functionally related to bacterial HASs. Considering the phylogenetic and biochemical connection between chitin synthases (CHSs), essential for fungal cell wall synthesis, and HASs, it is reasonable to hypothesize the latter might be more common in fungi than expected. In this work, a comprehensive in silico survey of putative HASs in the Fungal Tree of Life was carried out. 68 putative HASs, mainly in Basidiomycota, were found, although other AI-inferred HASs were found among Ascomycota. Global fold and arrangement of essential amino acids were shared by all kingdoms HASs; however, fungal HASs showed additional exclusive conserved sequence signatures. Moreover, fungal HASs bore an only 3-helices transmembranal pore and their gating loop, which regulates the entrance of substrates to the catalytic site, was directly connected to an also exclusive intrinsically disordered (IDR) C-terminus. Phylogenetically, fungal HASs were found in a clade different to that of bacterial, animal and viral HASs, and all HASs shared the same ancestor with class VI CHSs. The atypical features of fungal HASs could influence the size and biological role of the HA they synthesize and also highlight potential regulatory differences among HASs at the gating loop configuration level. ImportanceDespite the report of CPS1p, the hyaluronic acid synthase (HAS) of Cryptococcus neoformans, the diversity, structural features and biochemical assets of fungal HASs remain unknown. Here, 68 putative fungal HASs were identified, mainly among Basidiomycota. Although their fold is similar to that of already characterized HASs, their transmembranal pore, integrated by only 3 helices, and their atypical gating loop configuration, suggest they could be also differently regulated, influencing size and function of HA they synthesize.

Brain vascular aging is increasingly recognized as a critical therapeutic target for age-related cognitive decline. Oxidative stress, bioenergetic dysfunction, and molecular damage play central roles in the progression of vascular aging, contributing to cerebrovascular dysfunction and impaired cognitive function. While naturally occurring polyphenols such as resveratrol (RSV) have demonstrated potential in mitigating aging-related pathologies, their poor bioavailability and limited brain targeting efficiency significantly constrain their therapeutic impact. As a result, high doses or advanced drug delivery strategies are necessary to achieve meaningful physiological effects. We introduce a novel nanocarrier system designed to enhance RSV delivery to the cerebral endothelium by leveraging the natural formation of an apolipoprotein E (ApoE)-enriched protein corona around fusogenic liposomes (FL) in vivo. These nanoparticles directly fuse with cytoplasmic cell membranes and thus evade endocytosis. We found that once in the circulation FL spontaneously acquire a protein corona, which is highly enriched in ApoE, a key ligand for brain endothelial low-density lipoprotein receptors (LDLR). Based on this observation, we engineered an ApoE-functionalized protein corona around FL (ApoE-FL) to systematically evaluate whether this mechanism could be exploited for targeted brain delivery. Following optimization and physicochemical characterization, the RSV-loaded liposomes were evaluated in vitro using human cerebral microvascular endothelial cells and in vivo C57BL/6 aged mice to assess their therapeutic potential. Both FL and engineered ApoE-FL liposomal delivery systems exhibited a strong affinity for endothelial cell membranes in vitro. The knockdown of the ApoE receptor, low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced liposomal docking. Microscopy analysis revealed that both ApoE-FL and non-functionalized FL directly fused with endothelial plasma membranes, thus bypassing intracellular organelles and minimizing lysosomal degradation. This suggests that the naturally formed ApoE corona in vivo may contribute to efficient cerebrovascular targeting, a property successfully replicated by the engineered ApoE corona strategy. In vivo biodistribution and kinetic studies demonstrated that especially ApoE-FL achieved enhanced brain-targeting efficiency, prolonged cerebrovascular retention, and extended targeting distance along the arteriovenous axis. This emphasizes that fusogenic liposomes effectively engage almost the entire microvascular network, including capillaries and post-capillary venules. Functionally, fusogenic liposome-delivered RSV improved blood-brain barrier (BBB) integrity, enhanced neurovascular coupling (NVC) responses, and promoted brain vascularization in aged mice. Single-cell RNA sequencing (scRNA-seq) revealed enhanced endothelial angiogenesis and barrier protective transcriptional profiles in cerebrovascular cells treated with ApoE-FL/RSV, suggesting a molecular basis for the observed vascular benefits. Liposomal RSV delivery achieved near-complete cerebrovascular and cognitive rejuvenation in aged mice applying a 2000-fold lower RSV dose than oral administration used as control sample. Thus, ApoE-FL liposomes exhibited exceptionally high delivery efficiency in deeper brain regions, further expanding their therapeutic potential. These findings underscore the importance of targeted drug delivery in optimizing therapeutic outcomes and establish ApoE-functionalized fusogenic liposomes as a promising strategy for mitigating brain vascular aging and cognitive decline. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/709925v1_ufig1.gif" ALT="Figure 1000"> View larger version (52K): org.highwire.dtl.DTLVardef@f7966dorg.highwire.dtl.DTLVardef@b4ea4corg.highwire.dtl.DTLVardef@18240a9org.highwire.dtl.DTLVardef@634f6a_HPS_FORMAT_FIGEXP M_FIG C_FIG

This study presents a multidisciplinary approach to evaluate the structure formation and digestion of lupin protein crosslinked with transglutaminase (TG). TG was applied at 0-10 U/g protein, and structural development was assessed by oscillatory rheology (G, G"), while SDS-PAGE and o-phthaldialdehyde (OPA) assays were used to evaluate protein participation and the reduction of free {varepsilon}-amino groups, respectively. Proteomics was further employed to characterise molecular features associated with crosslinking behaviour. Lupin protein showed a clear dose-dependent increase in gel strength during incubation, with G values reaching 214 {+/-} 43.9 Pa at 10 U/g TG, compared to 7.2 {+/-} 0.6 Pa in the untreated control. Across all conditions, G remained higher than G" throughout frequency sweeps, and low tan {delta} values confirmed the formation of elastic networks driven by covalent crosslinks. SDS-PAGE and OPA results consistently demonstrated efficient crosslink formation, which increased with both incubation time and TG dosage, with SDS-PAGE indicating involvement of specific protein fractions. Proteomic analysis revealed disordered structural domains in the protein are preferred regions to form crosslinks. Furthermore, TG treatment was found to slow the digestibility of the crosslinked lupin protein. Overall, this work demonstrates how integrating proteomic insights with functional measurements can guide the selection and optimisation of plant proteins for enzymatic structuring. The approach offers a rational pathway to enhance the functionality of alternative protein sources such as lupin, supporting the development of sustainable food systems, including applications in meat and dairy analogues.

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind {beta}-1,3- and {beta}-1,6-glucans with high specificity. CpCBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the proteins binding sites plays distinct roles in ligand binding. One binding site, referred to as the {beta} site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on CpCBM92A. The other two binding sites, referred to as and {gamma}, preferentially bind longer segments of {beta}-1,3- and {beta}-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.

Heterotrophic marine microorganisms have the capability to degrade and metabolize laminarin, which is the most abundant source of energy and nutrients in the marine environment, via enzymes encoded by genes clustered in polysaccharide utilization loci (PULs). In this study, a PUL potentially responsible for laminarin utilization was identified in the genome of the marine bacterium Muricauda lutaonensis strain ISCAR-4703, with conserved synteny in the genus Muricauda. A GH17 laminarinase (MlGH17A) encoded in the newly identified PUL was cloned, produced, and characterized as an endo-acting laminarinase, exhibiting the ability to degrade laminarin and laminari-oligosaccharides with a degree of polymerization (DP) greater than four into laminaribiose, laminaritriose, and laminaritetraose, with laminaritriose as the main product making up >50% of the produced oligosaccharide products. The three-dimensional model of the enzyme revealed the presence of seven putative subsites, including four glycone subsites (-4 to -1) and three aglycone subsites (+1 to +3), with a wide cleft to accommodate branches at the -2 subsite, enabling it to act on {beta}-1,3 linked backbones in polysaccharides with {beta}-1,6 linked branches. This enzyme is, along with the recently characterized {beta}-1,3 glucanosyltransglycosylase (MlGH17B), conserved in several Muricauda species and is suggested to play a crucial role in the utilization of laminarin by these bacteria. ImportanceLaminarin, a {beta}-1,3-glucan with occasional {beta}-1,6 branching, is the most abundant source of energy and nutrients in the marine environment. In this study, polysaccharide utilization loci (PULs) for laminarin degradation were identified in various marine Muricauda species, encoding a range of glycoside hydrolases and transglycosylases. In Muricauda lutaonensis ISCAR-4703, the PUL included two GH17 enzymes, separated by a GH30 enzyme and a major facilitator superfamily (MFS) transporter, a feature observed in all corresponding Muricauda PULs. A novel endoacting laminarinase from the PUL, MlGH17A, was characterized and shown to hydrolyze laminarin into laminaribiose, laminaritriose, and laminaritetraose, with laminaritriose as main product. Bioinformatic analysis showed that the enzyme lacked the typical subdomain found in GH17 plant {beta}-glucanases, leading to a lower number of aglycone subsites (+1 to +3). Instead, MlGH17A possessed more glycone subsites (-1 to -4), attributed to the {beta}3-3 loop, which was longer than in GH17 plant {beta}-glucanases.

TBCK-related encephalopathy (TBCKE) is a neurodevelopmental disorder associated with biallelic mutations in TBCK. Despite the increasing number of reported cases worldwide, the biochemical and biophysical properties of TBCK remain unclear, hindering molecular understanding of its role in disease. Here, we present the successful expression, purification, and biochemical characterization of full-length human TBCK produced in Spodoptera frugiperda cells. Biochemical and biophysical analyses reveal that the catalytically inactive pseudokinase domain of TBCK lacks nucleotide binding, consistent with the absence of the canonical VAIK, HRD, and DFG motifs required for catalysis. These findings support that TBCK is a class I pseudokinase and provide a foundation for future structural and functional studies to elucidate its biological role.

Mutations in MAPT, the tau gene, give rise to forms of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T), with abundant filamentous tau inclusions in brain cells. Some mutations that encode missense and deletion variants can give rise to a clinical picture of Picks disease and filaments made of three-repeat tau. Here we report the electron cryo-microscopy (cryo-EM) structures of tau filaments from individuals with MAPT mutations D252V, G272V, S320F and {Delta}G389-I392. The two-layered Pick fold was present in the individuals with mutations D252V and {Delta}G389-I392. By contrast, mutations G272V and S320F gave rise to a more open variant of the Pick fold, with residues 272-341 rotated by 20-25{degrees} with respect to the rest of the structure. These findings show that missense mutations within the filament core can modify the Pick fold, generating closely related structural variants. In addition, we were able to reconstitute the Pick fold and some of its variants using seeded assembly with recombinant 0N3R tau carrying 12 serine or threonine to aspartate substitutions (PAD12) and missense mutations D252V, G272V or S320F. This work provides a foundation for the development of structure-based diagnostic and therapeutic approaches.

{beta}-mannans are plant structural and storage polysaccharides prevalent in the human diet. Their degradation in the gastrointestinal tract is mediated by the human gut microbiota (HGM) through expression of a plethora of carbohydrate-active enzymes (CAZymes), although our understanding of the details of mannan breakdown is lacking. In this study, a prominent HGM member, Bacteroides cellulosilyticus, was found to be exceptionally efficient at utilising {beta}-mannans, mediated by the expression of a single polysaccharide utilisation locus (PUL). Amongst the predicted surface CAZymes encoded in the PUL, we identified a family 26 glycoside hydrolase of an unusual molecular architecture. BcWH2_GH26 contains a putative carbohydrate-binding module (CBM) directly intercalated into its catalytic domain, unlike classical CBMs which are located at the N- or C-termini of the catalytic domain. Phylogenetic and functional analyses of this internal CBM, and a homologue from another mannan user Bacteroides uniformis, revealed a narrow specificity for {beta}-mannan and support their classification as a novel CBM family. To investigate the evolutionary basis for the unusual enzyme architecture, the effect of the CBM on the catalytic activity of the enzyme was assessed. No significant differences in the kinetic parameters were found between the full-length and CBM deletion constructs against both soluble and insoluble mannans. The potential role of the internal CBM in enzyme function is discussed in the context of the likely localisation of the BcWH2_GH26 in the outer membrane utilisome encoded by the Bc mannan PUL.

The intracellular transport system is pivotal for cellular function and integrity, facilitated by cytoskeletal motor proteins such as dynein, which traverse along microtubules (MTs). The heterogeneity of the tubulin isotypes composing MTs introduces functional diversity, potentially affecting cytoskeletal motor proteins interactions with the MT. This in silico study investigated the influence of amino acid sequence variations in the C-terminal tails (CTTs) of six different Homo sapiens tubulin isotypes, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4A, and TUBB5, highly expressed in human brain tumors, and assessed the isotypes effect on the binding of motor protein dynein to MT. Among these isotypes, TUBB2A, TUBB2B, and TUBB2C were found to affect conformational motions of the dyneins microtubule-binding domain (MTBD) and stalk domain. The investigation highlighted the novel role of isotype-specific variations in lateral interactions between tubulin protofilaments (PFs) in determining the proximity of the {beta}-CTT of the adjacent PF to the MTBD, potentially affecting dyneins motility and suggesting how changes in isotype expression directly influence dyneins velocity and processivity and contribute to transport defects associated with neurological disorders and cancers. Isolating specific tubulin isotypes experimentally is challenging due to their high sequence similarity and complex interactions with other microtubule-associated proteins. This makes it challenging to distinguish between different tubulin isotypes and their effects, particularly in tissues where multiple isotypes are co-expressed. Additionally, these isotypes are heavily modified in vivo by post-translational modifications, which further complicate the isolation of a single, unmodified tubulin isotype. As a result, computational approaches have been necessary in this study for exploring these effects in a controlled, isotype-specific manner.

Heavy metal ATPases (HMAs) are important group of transmembrane proteins involved in homeostasis of metal ions in plant systems. In this study, a comprehensive analysis of genome assembly (VC1973A v7.1) resulted in the identification of nine HMA genes (VrHMA) and their corresponding proteins in Mungbean, an agronomically important legume crop known for its nutritional values. VrHMA proteins were also characterized based on their biomolecular features, conserved domains and motifs arrangement, transmembrane helices, pore-line helices, subcellular location and occurrence of signal peptides. Based on sequence homology, nine VrHMAs were clustered into two major substrate-specific groups: VrHMA1, VrHMA5 and VrHMA7 were categorized under the Zn/Co/Cd/Pb ATPase group, whereas the remaining six VrHMAs belong to the Cu/Ag subgroup. Gene structure analysis and promoter scanning revealed the structural divergence and presence of various stress-responsive cis-acting elements, respectively. The expression analysis of VrHMA genes in root and leaf tissues, in response to heavy metal (Zn, Cd and Cu) stress, indicates their role in the uptake, transport and sequestration of metal ions. Interestingly, VrHMA5 showed incremental upregulation in roots in response to all three heavy metal stresses, whereas its expression was only upregulated in the leaf tissues under Zn stress, which indicates its role in vascular transport in V. radiata. In addition, this study provides valuable insights into the functional roles of VrHMA genes and will lay a foundation for future genetic improvement in mung bean aimed at enhanced heavy metal stress tolerance and micronutrient homeostasis.

Glucan-water-dikinase 1 (GWD1) plays an essential role in regulating starch metabolism in plants via O-6 phosphorylation of amylopectin. Here, we used biochemical characterization, AlphaFold2 modeling, X-ray crystallography and Small-Angle X-ray Scattering (SAXS) experiments to study its structure and catalytic mechanism. The protein is organized into five domains with two carbohydrate-binding modules (CBMs) at its N-terminal end followed by a central domain, whose structure was solved by X-ray crystallography in open and closed conformations. Next comes the domain carrying the catalytic histidine and the ATP-binding domain. We studied the spatial arrangement of the full enzyme and of several truncated forms by SAXS-driven modeling and identified a pivoting movement of the Histidine domain consistent with the enzymes autophosphorylation and subsequent phosphate transfer to a glucan. Our data suggest important residues at the domain interfaces that might assist catalysis and we hypothesize that the second CBM helps maintaining the catalytic domain close to the glucan chain for productive phosphate transfer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/704335v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1b860e5org.highwire.dtl.DTLVardef@1e172dcorg.highwire.dtl.DTLVardef@3c03edorg.highwire.dtl.DTLVardef@25c0d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

The role of vision in the behavior of blood-feeding mosquitoes has remained largely overlooked, particularly in species of the Anopheles genus, despite their significant impact on global health. While the importance of olfactory and thermal cues in host-seeking is well established, the contribution of visual stimuli to mating and feeding behavior remains far less understood. In particular, Anopheles mosquitoes exhibit complex swarming behavior that depends on visual input, suggesting an underexplored avenue of research with direct implications for vector control. This study introduces a genetically modified mosquito line lacking the enzyme Tan, a hydrolase involved in both dopamine and histamine metabolism, to investigate the behavioral relevance of visual cues in Anopheles. Through a combination of behavioral assays and controlled laboratory experiments, the impact of visual disruption on attraction to UV-B light, host-seeking, and blood-feeding success was assessed. The findings demonstrate a reduced light-dependent attraction in both Anopheles males and females, suggesting an impairment in visual processing or a related behavioral response. This observation has implications for reproductive success and potential adaptation to anthropogenic environments with artificial light. By leveraging this novel knockout model, the study offers new tools and perspectives to better understand how vision shapes mosquito behavior and how this knowledge could be harnessed in the development of next-generation vector control strategies.

Autophagy is a critical neuroprotective mechanism, the impairment of which can lead to severe neurodegenerative diseases. Spinocerebellar ataxia type 1 (SCA1) is a monogenic neurodegenerative disorder, characterised by the presence of protein aggregates and consequent loss of cellular functions. The expression of mutant Ataxin1 (ATXN1) in glial cells has been demonstrated to induce inflammatory responses and loss of supportive functions, thereby exacerbating neuronal degeneration in SCA1. Autophagic dysfunction has been shown to affect both neurons and glial cells, resulting in widespread pathological consequences. In this work, we aimed to evaluate the efficacy of two small-molecule autophagy activators, AUTEN-67 and AUTEN-99, in models of glia-specific SCA1 in Drosophila. Our results demonstrate that AUTEN-99 has a stronger autophagy enhancing effect, with significantly improved response times and survival rates, compared to untreated ATXN1 mutants. Glia-specific assays in mouse primary hippocampal cultures also confirmed that AUTEN-99 is a more effective activator. Ultimately, co-treatment of neuronal and glial cultures did not reveal any synergistic benefits from combining the two AUTEN compounds compared to single-agent treatment. Our findings contribute to a better understanding of the utility of AUTENs and may help to understand the critical role of autophagy in neurodegenerative diseases.

This work develops and characterises a hierachichal oral drug delivery system based on the microencpasulation of drug-loaded amphiphilic nanogels within a mucoadhesive alginate/chitosan shell. Results show a more controlled release and a statistically significant oral half-life with respect to the free drug.

Lacritin is an abundantly expressed glycoprotein in tear fluid and plays key roles in immune response, tear secretion, and bacterial killing. These biological functions are tightly regulated through several biochemical mechanisms including multimerization, proteolysis, and alternative splicing, especially within its C-terminal domain. Given its critical role at the ocular surface, lacritin is currently under investigation as a diagnostic biomarker and therapeutic candidate for dry eye disease (DED). However, despite over three decades since its initial discovery, the functional significance of the O-glycans that comprise more than 50% of its molecular weight remain largely unknown. To address this gap, we leveraged mass spectrometry (MS)-based glycoproteomics and molecular dynamics (MD) to explore the structural role of site-specific O-glycans on C-terminal lacritin. In doing do, we identified distinct glycosylation profiles between monomeric and multimeric lacritin, particularly at glycosites located near crosslinking residues (Lys101 and Lys104) that modulate multimer formation. Building on our glycoproteomics data, we performed MD simulations on monomer and multimer glycoforms and revealed that O-glycans participate in intra-glycan-protein interactions, thereby affecting the conformational flexibility of lacritin and the spatial arrangement of Lys101 and Lys104. Finally, we quantified the solvent-accessible surface area (SASA) of Lys101 and Lys104, highlighting that proximal O-glycosylation is predicted to affect the propensity of these residues to participate in crosslinking. Taken together, these findings underscore a central role for lacritin O-glycans in affecting structural topology with implications for its downstream biological activity.

Mycobacterium tuberculosis topoisomerase I (MtbTOP1) is essential for the viability of the causative agent of TB. There are still significant unanswered questions regarding the dynamic conformations during catalysis of relaxation of negatively supercoiled DNA by MtbTOP1. We aim to study the flexible hinge residues that control the dynamics of inter-domain rearrangements involved in the enzyme conformational changes that allow the opening-closing of the topoisomerase gate. We used the online server PACKMAN to predict possible hinges from the MtbTOP1 crystal structure. The predicted region "PRO506 to LEU526" at the border between domains D2 and D4 with a p-value <0.05 was then studied as a potential hinge. The highly conserved ARG516 from this region interacts with the DNA inside the protein toroidal cavity. This arginine maintains inter-domain interaction with GLU207 of D4 and ASP691 of D5 domains. After introducing alanine substitutions, we further studied the mutant topoisomerases in biochemical experiments. The results showed a significant loss in DNA relaxation activity without affecting DNA binding and cleavage after mutating GLU207 and ARG516, consistent with their role as hinge residues in domain rearrangements.

The ion channel KCa3.1 plays a role in immune regulation, red blood cell function, and is linked to numerous types of cancer. Various animal toxins, such as maurotoxin, bind to the extracellular side of KCa3.1, providing a potential starting point for inhibitor development. We report in this work the discovery of a novel, small-molecule inhibitor, with a micromolar IC50, which was specifically designed to target plasma-membrane KCa3.1 channels from the extracellular side. This compound can serve as a starting point for the development of more selective inhibitors and probes. For the identification of new extracellular inhibitors, molecular dynamics simulations were performed using the experimental structures of KCa3.1 and maurotoxin. The simulations produced a validated binding mode, highlighting key residues involved in the interaction between the toxin and the channel. These findings laid the foundation for the structure-based identification of novel extracellular small-molecule inhibitors of KCa3.1. The Molport database, containing approximately 50 million compounds, was screened using protein-ligand docking, yielding a hit molecule that was experimentally confirmed using patch clamp assays.

Carbohydrate-binding modules (CBMs) play crucial roles in carbohydrate-active enzymes by promoting substrate recognition and proximity, particularly for insoluble polysaccharides. Here, we report the discovery and characterization of a novel {beta}-trefoil structured CBM associated with a GH87 -1,3-glucanase from Flavobacterium johnsoniae, which accordingly was designated FjCBMXXXGH87. The full-length enzyme efficiently hydrolyzed -1,3-glucan (mutan) and -1,3/-1,6-glucan (alternan), whereas the catalytic domain alone displayed reduced activity, indicating that FjCBMXXXGH87 enhances substrate interaction. Pull-down assays confirmed that FjCBMXXXGH87 binds -1,3-linked glucans, and structural investigation together with site-directed mutagenesis identified two distinct binding sites essential for protein-ligand interactions. Phylogenetic analysis showed that CBMXXX homologs are present together with enzymes from families GH87, GH13, GH16, and GH99, and potentially may comprise up to three binding sites. Together, these findings establish FjCBMXXXGH87 as the founding member of a new CBM family, which may have broad functional versatility in polysaccharide recognition. This discovery expands the repertoire of {beta}-trefoil CBMs and provides new insights into carbohydrate recognition strategies relevant to -glucan degradation.

Staphylococcus aureus encounters massive oxidative stress during infection. To counter this, the bacterium developed robust antioxidative defense mechanism. Glutathione peroxidases (Gpx) are well characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. S. aureus possesses two putative Gpx genes but lacks GSH biosynthetic machinery and glutathione reductase required for canonical Gpx function, suggesting alternate electron donor system(s) may be involved. This study aimed to elucidate structure-based biochemical characterization of one of the S. aureus glutathione peroxidases homologs (SaGpx, Uniprot Id: Q2FYZ0) and identify its plausible electron donor system. Herein, we cloned, purified and determined the high-resolution crystal structure of SaGpx (1.5 [A] resolution) using X-ray diffraction crystallography. In vitro biochemical characterization of the highly conserved active site amino acid point mutants, as well as their structural disposition suggests their precise roles in the enzymes catalysis. The crystal structure of SaGpx revealed that the enzyme adopts a canonical glutathione peroxidase fold with conserved catalytic tetrad composed of C36, Q70, W124 and N125. Also, SaGpx shows similarity with mammalian Gpx4, which was previously shown to exert phospholipid hydroperoxide peroxidase activity. Furthermore, biochemical assays suggest that SaGpx utilizes Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism follows an atypical 2-cysteine peroxiredoxin-like pathway involving the formation of a sulfenic acid intermediate, followed by an intramolecular disulfide bond subsequently resolved by thioredoxin. This work provides the first structure-based biochemical characterization of a bacterial glutathione peroxidase homolog, establishing the novel structural insights of SaGpx as a noncanonical thioredoxin-dependent glutathione peroxidase.