International Journal of Biological Macromolecules

Drug targets and vaccines for bacterial diseases are challenging to develop. Computational subtractive genomics and reverse vaccinology offer a viable substitute for conventional drug and vaccine development approaches. In recent years, Pasteurella multocida, a Gram-negative pathogen responsible for severe infections in humans along with major diseases in livestock and poultry, has developed resistance to multiple drugs. As a result, there is an urgent demand for research into reliable, efficient, highly potent, and cost-effective strategies to combat the rise of Pasteurella multocida infections. To address this, the present study integrated an in silico subtractive genomics and reverse vaccinology workflow to pinpoint proteins with potential value as both drug and vaccine targets. From the complete Pasteurella multocida Pm70 proteome, we identified 446 proteins that had no similarities with human proteins, out of which 129 were found to be essential for the pathogens survival. These 129 proteins are therefore potential candidates for drug development. Further metabolic pathway analysis, virulence potential, subcellular localization, and structural analyses revealed two outer membrane proteins, namely, 47 kDa outer membrane protein and lipopolysaccharide-assembly protein LptD, as promising candidates for therapeutic development. Subsequent antigenicity analysis revealed both outer membrane proteins as potential vaccine targets, exhibiting highly favorable antigenicity scores. Candidate B- and T-cell epitopes were systematically mapped from these two targets based on antigenicity, immunogenicity, toxicity, and allergenicity potential and only high-confidence epitopes were assembled using adjuvants and class-specific linkers to construct the final multi-epitope vaccine (MEV). The designed MEV construct was demonstrated to be highly antigenic, non-toxic and non-allergenic to humans, while possessing favorable structural and physicochemical properties, and exhibited strong binding interactions with human toll-like receptors with minimal molecular deformability. Although further experimental validation is recommended to fully assess their efficacy, the present study establishes a solid foundation for the development of novel therapeutics and vaccine candidates against P. multocida, ultimately contributing to the reduction of its rising infections.

Carbohydrates are essential molecules involved in a wide range of biological processes. While their structural representation in the Protein Data Bank (PDB) has been hindered due to their highly diverse and flexible nature, advancements on X-ray crystallography and CryoEM are accelerating the obtaining of their experimental structures, which in turn supports improved analyses of their structural properties, such as the role of their non-chair conformations on biological processes. In this context, the current work provides a comprehensive and up to date overview of carbohydrate structures within the PDB through classification, quantification, and characterization of monosaccharides and glycan chains, incorporating Cremer-Pople puckering coordinates to evaluate pyranose conformations. Our findings show that, even with a steep increase in CryoEM structures including more complex oligosaccharides, carbohydrate data remains concentrate dinsmallglycanchains, with a about 4% of non-chair conformation sintopresolution structures. Free energy landscapes for the pyranose ring in the presence and absence of nine different binding proteins, including both enzymes and non-enzymes, reinforce the preferred stability of chairs and the rarity of conformational induction upon binding.

Microplastic pollution from homopolymers such as polypropylene (PP), polystyrene (PS), and polyethylene (PE) poses growing health and environmental concerns, highlighting the need for robust polymer-degrading biocatalysts. We previously identified a naturally occurring cysteine-dependent hydrolase (CsdH) from Rhodococcus opacus, featuring a unique Cys160-Lys127-Asp39 catalytic triad and providing a novel mechanism for polyethylene terephthalate (PET) degradation. In this study, we have determined crystal structures of CsdH in complex with PP, PS, PE, and styrene and demonstrated the potential of the enzyme to directly degrade these homopolymers into their respective low molecular weight degradation products using LC-MS. Structural analysis reveals that the enzyme retains a conserved principal access tunnel while undergoing subtle substrate-dependent changes to accommodate different substrates. The enzyme exhibited broad plastic degradation activity with the highest weight loss observed for PE, followed by PP and PS. SEM analysis reveals surface roughness, cracks and grooves, confirming polymer degradation. Further, these results corroborate through FTIR and TGA analysis. These findings establish a strong foundation for enzyme engineering and aim at developing efficient microplastic degradation.

Whether the homologous parallel triplex (HP-triplex, formed by dsDNA and a homologous ssDNA) acts as an intermediate prior to DNA recombination remains controversy, owing to the intrinsic instability and lake of experimental data. Here, we report a HP-triplex that is stable at least at 35 oC under protein-free conditions. Two complementary circular ssDNAs were first hybridized at a 1:1 ratio to form an LR-chimera containing both Z-DNA and B-DNA. Then the circular homologous ssDNA (T-rich) was found to easily hybridize with this LR-chimera to form the HP-triplex (strand ratio of 2:1), involving both left- and right-handed triplex parts under the topological constraint. The third strand locates in the major groove and forms hydrogen bonds with both bases in each base pair of dsDNA without obvious strand exchange. Our data indicate that the third-strand ssDNA may first bind to the B-DNA region, followed by association with the Z-DNA region to assemble the full triplex structure. Structural simulations and analyses reveal that topological constraint imposed by the circular architecture raises the energy barrier for strand displacement, thereby stabilizing this triplex. In summary, the dynamic HP triplex can be stabilized via a Z B junction structure that mimics the in vivo one formed by negative supercoiling. These findings demonstrate that HP-triplex may act as an intermediate for DNA recombination, anti-gene strategy, and other interactions between dsDNA and homologous ssDNA or RNA.

Antifreeze proteins and peptides adhere to ice crystals and inhibit their growth, resulting in freezing point depression. AFP6 is an abundant antifreeze peptide in the blood serum of winter flounder (Pseudopleuronectes americanus). This 37-residue peptide forms an amphiphilic -helix with a hydrophobic ice-binding surface. In this study, prediction of the peptide structure suggested the formation of an antiparallel dimer burying the hydrophobic surfaces of both subunits. The oligomerisation of AFP6 was therefore examined in aqueous solutions using size-exclusion chromatography and chemical crosslinking along with ice-binding evaluation. Crosslinking of non-amidated AFP6, followed by denaturing electrophoresis, revealed bands consistent with monomers and dimers, whereas results for the amidated form and a recombinant mutant suggested additional larger forms. Bands consistent with the presence of monomers and dimers were also predominant following non-amidated AFP6 crosslinking under a variety of solution conditions, with smaller proportions of peptide assembled up to tetrameric sizes; however, the larger oligomers appeared to be absent from AFP6 treated with sodium bicarbonate. Isolated oligomers of non-amidated AFP6 produced ice crystal morphologies and thermal hysteresis comparable to those for the unfractionated peptide suggesting similar interaction with ice. Oligomerisation of AFP6 may allow greater solubility of this amphiphilic -helical peptide in aqueous solutions.

Golgins are widely described as long coiled-coil proteins that contribute to the structural organisation and trafficking functions of the Golgi apparatus. Although experimental structures have been determined for a limited number of golgin regions, atomic-level information on their extended coiled-coil segments remains scarce, and the oligomeric state, topology, and register of most predicted regions remain unestablished. Here, we characterise a predicted coiled-coil region of the yeast golgin Bug1 (BUG1cc) using structural, biophysical, and computational approaches. X-ray crystallography revealed a parallel, in-register dimeric coiled-coil containing ten heptad repeats and a predominantly hydrophobic core, with specific polar interactions contributing to dimer stabilisation. In solution, BUG1cc was dimeric under SEC-MALS conditions and remained highly alpha-helical across the pH and ionic strength conditions examined. CD measurements revealed pronounced scan-rate-dependent hysteresis, while DSC independently confirmed an asymmetry between heating and cooling transitions. Increasing protein concentration shifted both apparent transition temperatures while preserving thermal hysteresis, supporting chain association and conformational rearrangements in structural recovery. Structure-based simulations indicated that interface contacts and intra-chain helicity are thermodynamically coupled and melt as a single cooperative unit, and that the monomer released on dissociation is compact and only partially helical, so that reassociation proceeds through a coupled folding-binding mechanism whose rate-limiting step is conformational rather than bimolecular. Together, these results establish the molecular architecture of a predicted coiled-coil region of Bug1 and reveal a complex folding landscape in which oligomerisation, secondary-structure recovery, and kinetic barriers are tightly coupled.

Liquid-liquid phase separation (LLPS) of biomacromolecules has been shown to influence a large variety of biological processes via the formation of membraneless organelles. Aberrant LLPS, however, has been implicated in a number of oncological and neurodegenerative disorders. Proteins related to Alzheimer's, Parkinson's diseases and other major amyloidoses have all been shown to form these condensates. Interestingly, some amyloidogenic proteins were demonstrated to form heterotypic droplets, where both molecules coexisted within the same condensate and affected each other's aggregation. While such cross-interactions have been reported numerous times under non-LLPS conditions, the heterotypic droplet assemblies were only shown for a very limited number of amyloidogenic proteins. In this work, we examine heterotypic LLPS of prion proteins and the pro-inflammatory S100A9, whose cross-interaction under non-LLPS conditions we confirmed in a previous work. The results show that this interaction under condensation-promoting conditions enhances their aggregation, leading to the formation of a higher concentration of amyloid structures. These findings suggest that the pro-inflammatory S100A9 may have a critical role in promoting prion protein aggregation via the assembly of heterotypic condensates under high molecular crowding conditions.

The cornea is the outermost layer of the eye, most vulnerable to injury including from chemical exposure, either environmental, industrial or chemical weapons. For example, exposure of the eye to sulfur mustard (SM) can cause mustard gas keratopathy, resulting in devastating consequences for vision. Available treatments for corneal mustard keratopathy are either ineffective, or if used for a prolonged time, have deleterious side effects. Here, we aimed to harness the anti-inflammatory and anti-angiogenic peptide angiotensin-(1-7), known as Ang-(1-7), that is derived from the renin-angiotensin system (RAS). We designed and developed a protein-like polymer (PLP) displaying a brush of norleucine modified Ang (1-7) peptides (NLE Ang (1-7) PLP), which is a more potent derivative of Ang-(1-7), for administration as an eye drop. In a mouse model of nitrogen mustard (NM)-induced corneal injury, topical treatment and intraperitoneal (i.p.) injection of NLE Ang (1-7) PLP persistently minimized: (i) corneal haze; (ii) edema; (iii), expression of pro-inflammatory and pro-angiogenic genes; and (iv) neovascularization and conjunctivalization as evidenced after a prolonged treatment and recovery period. Collectively, our observations have demonstrated the efficacy of this novel polymer proteomimetic based eye drop treatment in reducing chemical agent-induced corneal injury as well as indicating its potential application in other ocular surface diseases associated with inflammation and angiogenesis.

Gastrointestinal infections are a leading cause of death and morbidity worldwide. Antibiotics as current treatments are ineffective or not recommended due to the emergence of multidrug resistant organisms. The initial step of the pathogenesis for most of the enteric infections is the binding of the pathogen to oligosaccharide receptors on the surface of host cells, making these receptors attractive targets for novel antimicrobial strategies that do not rely on conventional antibiotics. Here, we developed an alternative therapeutic intervention based on a novel microbiome-based chemical engineering approach. We synthesized cholesteryl-peptides as flexible chemical scaffold for the presentation of carbohydrate GM1 mimic receptors in probiotic bacteria acting as a decoy for cholera toxin neutralization. The GM1-cholesteryl-peptides were stably embedded into the membranes of both gram positive and gram negative probiotics, Lactobacillus reuteri DSM and Escherichia coli Nissle 1917 respectively. The resulting GM1-coated probiotics demonstrated to quench nearly 100% of cholera toxin in vitro, functioning as decoys by preventing toxin binding to epithelial cells. The present study strongly suggests that our new and versatile biomimetic non-recombinant system could be employed to neutralize the functional effect of infections mediated by oligosaccharide receptors.

Thrombostasin is a cysteine-free anticoagulant protein produced by the horn fly Haematobia irritans, featuring an unusual stretch of seven consecutive aspartate residues within the polypeptide sequence. Here, we report the first chemical synthesis of native thrombostasin and a hepta-glutamate analogue, together with assessment of their thrombin inhibitory and anticoagulant activity.

Although use of AI-based tools has gained ground in molecular biology research, their reliability remains a matter of debate. Here, we combined AlphaFold 3 modelling with microscale thermophoresis to investigate the interactions between glucan-binding protein C and the biofilm-inhibiting aptamer and its five derivatives. Structural modelling identified a binding motif on the aptamer and suggested a mechanism of action; however, the predictions were only partially supported by the experimental results. The AlphaFold generated confidence metrics did not correlate directly with the measured binding affinities, indicating that confidence scores can help identify plausible interaction interfaces, but they are not reliable predictors of binding strength. These results highlight the importance of integrating structural prediction with biophysical validation to dissect aptamer-protein recognition.

Metacaspases are cysteine proteases and are found in every group of organisms except animals. They are structural orthologs of caspases, which orchestrate apoptosis in animals. Caspases, metacaspases, paracaspases and orthocaspases are class C14 proteases with a similar active site (the hemoglobinase fold) and a catalytic cysteine residue. However, metacaspases cleave after arginine and lysine instead of aspartate and some researchers point out that a name ending with caspase is unsuitable. Also, most metacaspases are activated by calcium, they do not target the same proteins as caspases and they cut multiply instead of once. The Candida albicans metacaspase Mca1p mediates cell death in response to various stresses but also has pro-life functions. Many plant and protist metacaspases lack cell death roles and are involved in development, differentiation and immunity. This article highlights an interesting phenomenon, whereby the promotion of stress sensitivity and reduction of virulence, by Mca1p in C. albicans cells from exponential culture are abolished and reversed, respectively in cells from stationary-phase culture. This hints at the proteases's dual nature and at the conditions that might initiate a switch between pro-life and pro-death functions.

Structure-aware proteolysis prediction often ranks candidate cleavage sites using solvent accessibility from static protein structures, yet this assumption is rarely tested against native-state cleavage data. We examined this problem using AprX, a serralysin-family protease that readily hydrolyses caseins but shows lower activity toward native whey proteins. Across six bovine milk proteins, sequence compatibility and local scissile-bond accessibility did not separate caseins from whey proteins. In contrast, accessibility fell progressively in whey proteins as the required contiguous substrate window increased, reaching 0% across the working P4-P4' footprint, while all evaluable casein windows remained accessible in isolated-chain ensembles. Local remodelling showed that whey segments could still adopt AprX-compatible backbone geometry. An external thermolysin benchmark rejected a rigid whole-protein clash metric, but did not alter the contiguous-accessibility result. These findings identify footprint-scale accessibility as a distinct structural variable and show why single-residue solvent exposure can overestimate protease accessibility in folded proteins.

Numerous CRISPR-Cas systems have been developed for molecular detection of genetic elements exploiting the trans-cleavage activity of LbCas12a combined with short fluorescent probes. Alongside, a large variability of buffer conditions has been reported. However, how solution chemistry balances enzymatic turnover, signal stability, and detection performance have not been systematically defined. Here, we evaluated how the interplay between anions (chloride vs. acetate), additives, sequence probes, and ionic strength contributes to LbCas12a trans-cleavage activity. Buffer composition screening revealed that chloride-based buffers maintain a low background but slow catalytic rates, meanwhile acetate-based buffers accelerate enzymatic turnover with and without the target DNA sequence, increasing non-specific signals. Reducing agents and surfactants were the best enhancer combinations, improving cleavage performance by increasing ternary complex formation (Km). High salt concentrations only slightly improved the reaction kinetics. Finally, the 5FAM-TTATT-3BHQ-1 reporter showed the best balance between signal intensity and nonspecific background. Our findings indicate that LbCas12a tolerates a vast buffer composition landscape, maintaining robust trans-cleavage activity and detection performance. However, conditions that enhance catalytic activity simultaneously increase nonspecific probe cleavage and higher background, resulting in lower detection performance. Therefore, assay optimization should prioritize detection performance rather than maximal fluorescence output, where solution chemistry is a critical determinant.

[FeFe]-hydrogenases are molecular hydrogen (H2) converting enzymes that employ a hexanuclear iron-complex, the H-cluster, as catalytic cofactor, and a proton transfer pathway (PTP) that allows efficient proton coupled electron transfer (PCET). Recent phylogenetic analyses revealed different groups of [FeFe]-hydrogenases. Although only very few members from other groups have been characterized, current knowledge suggests high catalytic activity is predominantly associated with group A members. Here, we show that metal ions inhibit group A [FeFe]-hydrogenases by binding to three specific residues at the entrance of the PTP. Exchanging residue H565 of Clostridium pasteurianum CpI results in a metal-insensitive protein variant with wildtype like activity. In contrast, exchanging S320 and H569 in CpI, and their counterparts in additional group A [FeFe]-hydrogenases results in enzymes with strongly decreased activities and large overpotential requirements. These features are consistent with important roles of both residues in catalytic proton transfer. CpI structures reveal that H569, locally anchored by E278, can be present in two conformations so that we propose a histidine-dependent pendulum mechanism for exchanging protons between bulk solvent and the PTP, which is well supported by theoretical calculations. By that, our study widens information on how the mobility of histidine, governed by residues in the vicinity, contributes to the fundamental concept of PCET.

Glucoamylase is essential for industrial starch saccharification, but the limited thermostability and near-neutral pH tolerance of fungal glucoamylases necessitate cooling and acidification of liquefied starch. Here, we developed an artificial intelligence-guided strategy to simultaneously improve the thermostability, pH tolerance, and catalytic activity of glucoamylase from Penicillium oxalicum (PoGA). Two property-specific machine-learning models, CASPE-T and CASPE-A, identified substitutions associated with thermostability and pH tolerance, respectively. Experimental screening identified beneficial substitutions in 11 of 21 CASPE-T and 12 of 22 CASPE-A candidates. Folding-energy-guided recombination integrated the two traits while maintaining structural compatibility. The optimal variant, PoGA T513E/Q305N, exhibited 2.21-fold higher specific activity than the wild type, with half-life extended from 22.3 to 57.9 min at 60 degrees C and from 16.6 to 64.7 min at pH 8.0. Molecular dynamics simulations attributed these improvements to reinforcement of high-occupancy hydrogen-bonding networks, suppression of conformational fluctuations in the linker and carbohydrate-binding module, enhanced long-range dynamic coordination, and preservation of a compact catalytic architecture. At 60 degrees C and pH 6.5 without acidification, PoGA T513E/Q305N produced 219.9 g/L glucose and achieved 89.1% starch conversion, 31.4% higher than the wild type. This work provides an efficient framework for multi-objective enzyme engineering and sustainable starch biorefining.

The sulfur mobilization (SUF) system is the only Fe-S cluster biosynthesis pathway in Mycobacterium tuberculosis (M. tb) and Mycolicibacterium smegmatis (M. smegmatis). In this pathway, SufC protein acts as an ATPase in the SufBC2D scaffold for Fe-S cluster biogenesis. This crucial role positions SufC as a promising target for the development of antimycobacterial therapeutics. However, the SufC ATPases from Mycobacterial species are not yet characterized. This study includes cloning, expression, purification, and characterization of SufC homologs from M. tb (Rv1463) and M. smegmatis (MSMEG_3124) using biochemical, biophysical, and computational approaches. Both SufC proteins were purified using affinity and size-exclusion chromatography and observed to exist as monomers in solution. Although both proteins have the conserved domains, they exhibit different ATP-dependent kinetic efficiencies, demonstrating differences in their local environment and catalytic behavior. Furthermore, CRISPR interference targeting MSMEG_3124 results led to diminished M. smegmatis growth, highlighting the critical role of SufC in cellular viability. Modeling and virtual screening led to the identification of three candidate compounds, of which Z2911048515 and Z66741142 were selected for experimental evaluation. Circular dichroism spectroscopy analysis revealed slight differences in the spectral features of Rv1463 in the presence of Z2911048515 and Z66741142, while significant spectral variations were observed for MSMEG_3124. Importantly, Z66741142 was able to cause changes in the fluorescence spectra of both SufC protein homologs. Surprisingly, both compounds showed only limited inhibition of SufC ATPase activity, exhibited distinct modes of action, and may serve as preliminary chemical scaffolds for optimization and testing as effective inhibitors. This study presents functional diversity of mycobacterial SufC proteins across pathogenic and non-pathogenic species and provides a basis for developing a framework for further mechanistic and structure-guided investigation within the indispensable SUF pathway.

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of the ABC-core of SUR2B. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular KATP channels.

The development of NIR fluorescent dyes with favorable optical properties is of great importance especially for applications within the field of optical imaging. In this study, we report the design, synthesis and photophysical characterization of novel BODIPY-Xanthene hybrid dyes obtained via Knoevenagel condensation between an asymmetrical BODIPY core and the functionalized xanthene aldehydes intermediates. The synthesized dyes ex-hibit absorption (667 nm) and emission bands (720-722 nm) within the NIR region. Detailed UV-Vis and fluo-rescence studies were performed and molar extinction coefficients and stokes shifts also calculated. Moreover, findings highlighting the influence of structural modifications on the optical behavior of the hybrid dyes and their potential utility in optical imaging applications are reported.

Calcium (Ca2+) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca2+; signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani, affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani-infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca2+; sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca2+, and the Ca2+-binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca2+ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca2+ and undergo minimal secondary and tertiary structural changes upon Ca2+-binding, in contrast to several Ca2+-binding proteins. Of note, SlCBL1-2 binds Ca2+ with apparent KD values of 526 {+/-} 113 {micro}M and 28.7 {+/-} 9.47 {micro}M, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca2+-binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca2+ sensor in defense signalling.