International Journal of Biological Macromolecules

Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focused on the proteins belonging to the secretory family of tardigrade proteins termed secreted-abundant heat soluble ("SAHS") proteins, and investigated their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevented desiccated liposomes from fusion, and enhanced desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the potential application of SAHS proteins as stabilizing molecules for preservation of cells.

Barnacles are typical fouling organisms which strongly adhere to immersed solid substrates by secreting proteinaceous adhesives called cement proteins (CPs). The self-assembly of the cement proteins forms a permanently bounded layer that binds barnacle to foreign surfaces. However, due to the abundance of cysteines in whole-length CP20, it is difficult to determine its natural structure and to properly describe its self-assembly properties. In this study, a putative functional motif of Balanus albicostatus CP20 (BalCP20) is identified and found to present distinctive self-assembly and wet-adhesion characteristics. The atomic force microscopy (AFM) and transmission electron microscope (TEM) investigations show that wildtype BalCP20-P3 forms grain-like spindles, which further assembly into fractal-like structures looks like ears of wheat. SDS-PAGE, AFM and LSCM show that DTT treatment opens up disulfide bonds between cysteines and disrupts the fractal-like structures (eras of wheat). Additionally, these morphologies are abolished when one of the BalCP20-P3 four cysteines is mutated by alanine. Circular dichroism (CD) results further suggest that the morphological diversity among BalCP20-P3 and its mutations lays on the proportion of -helix. The above results demonstrate that cysteines and disulfide bonds play a crucial role in the self-assembly of BalCP20-P3. This study provides new insights into BalCP20 underwater adhesion, and brings in new inspirations for the development of novel bionic underwater adhesive.

The shortened L-asparaginases half-life in leukemia patients due to elevated serum proteases, poses a challenge. This study aimed to enhance the stability of Halomonas elongata L-asparaginase against trypsin. Employing the trRosetta server, we modeled the enzymes 3D structure with a quality score of 96.5, revealing predominant secondary structure of random coils (42%), alpha helices (33%), and extended strands (20%) organized in two domains. Molecular docking unveiled a triad alignment among residues Thr16, Ser65, and Asp97 with L-asparagine. Site selection for mutation considered secondary structure prediction, dimerization analysis, trypsin cleavage site determination and epitope mapping. A library of enzyme variants was constructed through site saturation mutagenesis which led to the identification of the Arg206 to Thr, resulting in a 1.7-fold increased enzyme-specific activity (2400 U/mg) and heightened trypsin resistance. The mutant displayed a half-life of 3.47 hin human serum, approximately 50% longer than the wild type. In silico analyses confirmed structural stability, reduced flexibility, and enhanced substrate binding, contributing to increased proteolysis resistance and enzymatic activity. The Arg206Thr mutant exhibited anti-proliferative activity (IC50 of 1.45 U/ml) on leukemia cell line K562, suggesting potential therapeutic implications.

AbstractZearalenone hydrolase 101 (ZHD101), derived from Clonostachys rosea, is known to effectively degrade the main contaminant (zearalenone) in animal feed, but the thermal instability of ZHD101 limits its industrial application. In this study, we successfully enhanced the thermal stability of ZHD101 through two iterative rounds of rational molecular design. First, after the prediction of disulfide bond sites, ZHD101T229C was obtained, and a new disulfide bond was formed between two single ZHD101 subunits to construct a dimer ZHD101. Then, based on ZHD101T229C, two high-vibration hotspot amino acids N137 and D170 were selected by analyzing atomic position fluctuations and dynamic information A small and precise mutant library containing three mutants (ZHD101T229C/N137L, ZHD101T229C/D170L, and ZHD101T229C/D170C) was obtained by saturation mutagenesis and calculation of binding energy in silico. Compared with the wild type, the thermal half-inactivation temperature (T50) of ZHD101T229C/D170C increased by 7{degrees}C, its half-life (t1/2) increased by 200% at 50{degrees}C, and its melting temperature (Tm) increased by 18.1{degrees}C. Molecular docking suggested that new covalent bond formation and shorter bond distance may contribute to the improved thermal stability of ZHD101. The rational design strategy proposed in this work can provide a reference for the thermal stability modification and optimization of other proteins. ImportanceZearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin that poses a significant threat to animal feed safety. ZEN hydrolase 101 (ZHD101) catalyzes the conversion of ZEN into a nontoxic product, offering a promising detoxification strategy. However, the thermal instability of ZHD101 severely limits its industrial application. In this study, the thermal stability of ZHD101 was significantly improved through two rounds of rational design, resulting in the ZHD101T229C/D170C mutant. ZHD101T229C/D170C exhibited the improved half-inactivation temperature (T50), half-life (t1/2) and the highest melting temperature (Tm) reported thus far. Overall, the iterative combinatiorial mutation strategy involved the introduction of a disulfide bond between two single subunits and the application of B-factor analysis to identify hotspot amino acids residues. This approach effectively enhanced the enzymes structural stability, paving the way for its industrial application. ZHD101T229C/D170C and the rational design strategy presented in this work provide a robust framework for the thermal stability optimization of similar biocatalysts, advancing the practical use of ZHD101 in the feed industry.

The contribution of redox active properties of cysteines in intrinsically disordered regions (IDRs) of proteins is not very well acknowledged. Despite of providing structural stability and rigidity, intrinsically disordered cysteines are exceptional redox sensors and the redox status of the protein defines its structure. Experimental evidence suggests that the conformational heterogeneity of cysteines in intrinsically disordered proteins (IDPs) is related to numerous functions including regulation, structural changes and fuzzy complex formation. The unusual plasticity of IDPs make them suitable candidate to interact with many clients under specific conditions. Binding capabilities, dimerization and folding or unfolding nature of IDPs upon interaction with multiple clients assign distinct conformational changes associated with disulfide formation. Here we are going to focus on redox activity of IDPs, their dramatic roles that are not only restricted to cellular redox homeostasis and signaling pathways but also provide antioxidant, anti-apoptotic, binding and interactive power.

Pseudomonas aeruginosa SuhB (PaSuhB) is a member of the bacterial Inositol monophosphatase family proteins. Numerous scientific evidences suggest PaSuhB is the pleotropic regulator of different metabolic pathways involved in bacterial biofilm formation and virulence determination. In this study, we have solved the high-resolution crystal structures of PaSuhB in its apo and substrate (Inositol monophosphatase) bound forms. Moreover, we carried out 3D pharmacophore modelling of the bound substrate to identify gallic acid, a phyto-phenol, abundant in medicinal plants, as a novel PaSuhB inhibitor. The high-resolution crystal structure of gallic acid/PaSuhB binary complex leads to the identification of a novel allosteric ligand binding site of the protein. In vitro, gallic acid induces the cold-sensitive growth of P. aeruginosa and E. coli, the previously reported phenomenon observed in suhB deletion mutants and also inhibits the swimming motility of P. aeruginosa. The plausible anti-bacterial molecular mechanism of action of gallic acid is presented herein.

We have adapted split GFP technology into the protein nanoparticle platform, Vaccine Delivery system X (VADEX), created in previous study. To evaluate the capability of this new platform, a model protein, maltose binding protein (MBP), was fused to the {beta}-strand 11 of sfGFP and co-expressed with VADEX-10 which was composed of LYRRLE peptide and N-terminal part up to {beta}-strand 10 of sfGFP. When these two fusion proteins were expressed in a cell, they were assembled into PNP spontaneously with a dynamic light scattering (DLS) particle size of 26 nm. This nanoparticle platform was renamed as VADEX-Pro for its capacity of expressing large protein on PNP. The thermostability of the assembled PNP was verified by both SDS-PAGE and DLS analysis following treatment. This PNP was stable at 25 {degrees}C and at temperatures as high as 40 {degrees}C for at least two months. Mutations that replaced cysteine residue of the LYRRLE peptide with serine or alanine destabilized and induced degradation of the VADEX-based PNP. The results in this study showed that the non-covalent complementation of split sfGFP became irreversible when reconstituted sfGFP was assembled in a VADEX-Pro PNP. This platform may be applied in developing thermostable vaccines.

Siderophore-mediated iron acquisition is essential for the virulence of Aspergillus fumigatus, a fungus causing life-threatening aspergillosis. Developing drugs targeting the siderophore biosynthetic pathway could help improve disease management. The transacetylases SidF and SidL generate intermediates for different siderophores in A. fumigatus. A. fumigatus has a yet unidentified transacetylase that complements SidL during iron deficiency in SidL-lacking mutants. We present the first X-ray structure of SidF, revealing a conserved two-domain architecture with tetrameric assembly. Importantly, the N-terminal domain contributes to protein solubility and oligomerization, while the C-terminal domain containing the GCN5-related N-acetyltransferase (GNAT) motif is crucial for the enzymatic activity and mediates oligomer formation. Notably, AlphaFold modelling demonstrated structural similarity between SidF and SidL. Enzymatic assays showed that SidF can utilize acetyl-CoA as a donor, previously thought to be a substrate of SidL but not SidF, and selectively uses N5-hydroxy-L-ornithine as an acceptor. Based on these findings, we propose SidF as the unknown transacetylase complementing SidL activity, highlighting its central role in A. fumigatus siderophore biosynthesis. This study elucidates the structure of SidF and reveals a novel role in siderophore biosynthesis. Investigation of this uncharacterized GNAT protein enhances our understanding of fungal virulence and holds promise for its potential application in developing antifungal therapies.

Xylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 [A]. XynA is characterized with a classic (/{beta})8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

Mycobacterium tuberculosis uses the ESAT-6 system-1/type VII (ESX-1) system for secretion of virulence proteins into the host cell, however the mechanism of virulence proteins secretion, molecular components and regulation of ESX-1 system are only partly understood. In the current study, we have analyzed the biological function and recognition mechanism between ESX-1 virulence EspC and EccA1 ATPase proteins. The EspC enters into A549 human lung carcinoma cells and exhibited cytotoxicity, as observed in MTT Assay. To understand the recognition mechanism between EspC and EccA1 ATPase, the EspC and EccA1 mutants were generated based on EspC~EccA1 interactions, as observed in molecular modeling. Binding analysis shows that EspC export arm interacts specifically to the {beta}-hairpin insertion motif of the TPR domain of EccA1 ATPase. Mutations in these epitopes lead to significant decrease/or abolish the binding between EspC and EccA1 ATPase. Our study provides insight into biological function and recognition mechanism between EspC and EccA1 ATPase, which can be used as target to prevent EspC secretion/ or in general virulence factor secretion by mycobacterial ESX-1 system.

This study provides a quantitative and comprehensive analysis of 18 Methuselah (mth) protein variants from fruit flies, focusing on their evolutionary relationships, structural features, and functional roles in aging and longevity. Phylogenetic analysis identified two major clades of mth proteins, with the first clade indicating conserved functions across Drosophila species and the second clade reflecting gene duplication and diversification. The study found five distinct functional subclasses of mth proteins through amino acid frequency and poly-string analyses, linked to their structural diversity and role in longevity. Structural topology and post-translational modifications reveal similarities with G-protein-coupled receptors (GPCRs), suggesting that mth proteins are crucial for signal transduction and cellular health. Variability in propeptide cleavage sites and intrinsic protein disorder further highlight adaptive roles in signaling. The findings underscore the importance of a quantitative and comprehensive approach to studying Methuselah genes, offering insights into their functional versatility and evolutionary dynamics. This enhanced quantitative understanding contributes to advancing research on aging and longevity.

CRM1 is an important drug target in diseases such as cancer and viral infection. Plumbagin and oridonin, the herbal ingredients with known anti-cancer activities, were reported to inhibit CRM1-mediated nuclear export. However, their modes of CRM1 inhibition are unclear. Here, a multi-mutant of yeast CRM1 was engineered to enable the crystallization of these two small molecules in CRM1s NES-binding groove. Each structure showed three inhibitor-binding sites, among which two are conserved in humans. Besides the known binding site, another site also participated in oridonin and plumbagins CRM1 inhibition. While the plumbagin-bound NES groove resembled the NES-bound groove state, the oridonin-bound groove revealed for the first time a more open NES groove, which may potentially improve cargo-loading through a capture-and-tighten mechanism. Our work thus provides a tool for CRM1 inhibitor crystallization, new insights of CRM1-cargo interaction, and a structural basis for further development of these or other CRM1 inhibitors.

Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by {beta}-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae, a plant pathogen. We first solved the crystal structure of VdPelB at 1.2[A] resolution showing that it is a right-handed parallel {beta}-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. VdPelB was most active on non-methylesterified pectins, at pH 8 in presence of Ca2+ ions. VdPelB-G125R mutant was most active at pH 9 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.

Cofilin-2 is a member of the ADF/cofilin family, expressed extensively in adult muscle cells and involved in muscle maintenance and regeneration. Phosphorylated cofilin-2 is found in pre-fibrillar aggregates formed during idiopathic dilated cardiomyopathy. A recent study shows that phosphorylated cofilin-2, under oxidative distress, forms fibrillar aggregates. However, it remains unknown if cofilin-2 holds an innate propensity to form amyloid-like structures. In the present study, we employed various computational and biochemical techniques to explore the amyloid-forming potential of cofilin-2. We report that cofilin-2 possesses aggregation-prone regions (APRs), and these APRs get exposed to the surface, become solvent-accessible, and are involved in the intermolecular interactions during dimerization, an early stage of aggregation. Furthermore, the cofilin-2 amyloids, formed under physiological conditions, are capable of cross-seeding other monomeric globular proteins and amino acids, thus promoting their aggregation. We further show that Cys-39 and Cys-80 are critical in maintaining the thermodynamic stability of cofilin-2. The destabilizing effect of oxidation at Cys-39 but not that at Cys-80 is mitigated by Ser-3 phosphorylation. Cysteine oxidation leads to partial unfolding and loss of structure, suggesting that cysteine oxidation further induces early events of cofilin-2 aggregation. Overall, our results pose a possibility that cofilin-2 amyloidogenesis might be involved in the pathophysiology of diseases, such as myopathies. We propose that the exposure of APRs to the surface could provide mechanistic insight into the higher-order aggregation and amyloidogenesis of cofilin-2. Moreover, the cross-seeding activity of cofilin-2 amyloids hints towards its involvement in the hetero-aggregation in various amyloid-linked diseases.

External stress can disrupt protein homeostasis in organisms, necessitating the involvement of heat shock proteins (Hsps) to restore equilibrium and ensure survival. Unlike other organisms, the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius lacks Hsp100, Hsp90, and Hsp70, possessing only two small heat shock proteins (Hsp14 and Hsp20) and one group II chaperonin, Hsp60. This raises questions about how protein substrates are protected and transferred to Hsp60 for refolding without other chaperones. Our study focused on ATP-dependent Hsp60 in S. acidocaldarius, revealing its formation of oligomeric structures in the presence of ATP. While ATP hydrolysis is not essential for oligomer formation and lid closure, it is crucial for Hsp60s chaperone activity, effectively folding stress-denatured substrate proteins by stabilizing their folded conformations. The mechanism involves hydrophobic recognition of unfolded substrates, encapsulating and releasing them in a more folded state. Negatively charged inner surface of the ring seems to be responsible for driving the folding of the substrate. Importantly, Hsp14 was found to transfer substrate proteins to Hsp60{beta}, orchestrating their refolding into an active state. Beyond protein folding, Hsp60{beta} protects the membrane under stress, contributing to maintaining membrane rigidity. Hsp60 exhibits nested cooperativity in ATPase activity, adapting to ATP concentration changes and interestingly Hsp60{beta} and Hsp60{beta} complex shows a mosaic behaviour during ATP hydrolysis belonging to both group I and group II chaperonin respectively. In conclusion, our study provides insights into the intricate mechanisms employed by Hsp60 in S. acidocaldarius to maintain protein homeostasis. It offers a comprehensive understanding of Hsp60s role in the heat shock response pathway, shedding light on fundamental cellular processes in extremophilic archaea.

The double plant homeodomain fingers 3a (DPF3 isoform a) is a human epigenetic regulator involved in chromatin remodelling, cell division, and ciliogenesis. Most notably, this protein is deregulated in various cancer types and neurodegenerative diseases. In our previous work, the disorder nature of DPF3a, as well as its propensity to aggregate into amyloid fibrils, have been highlighted, making it an amyloidogenic intrinsically disordered protein (IDP). Due to their high chain accessibility, IDPs structure and function are modulated by phosphorylation. It has been reported that phosphorylation of DPF3a at S348 (pS348) by the casein kinase 2 (CK2) is implicated in cardiac hypertrophy. CK2 can also phosphorylate DPF3a at S138 (pS138), which is also located in an intrinsically disordered region (IDR). However, no structural information is available on phosphorylated DPF3a. In the present study, we investigated the effect of phosphorylation on DPF3a structural and aggregation properties. Two single-mutated phosphomimetics (S138E and S348E) were characterised in vitro and compared to DPF3a WT, while in silico analyses were performed on pS138 and pS348 to assess structural changes at the molecular level. Circular dichroism and fluorescence spectroscopy revealed that both phosphomimetics are hybrid IDPs, with increased turn and antiparallel {beta}-sheet content as well as more buried aromatic residues compared to DPF3a WT, suggesting conformational rearrangements and a more folded N-terminal region. In silico characterisation supported these results, showing that phosphorylation of S138 and S348 induce extended conformation, especially the C-terminal extremity, due to electrostatic repulsion, while local folding occurs due to a proximity with arginine and lysine residues. Furthermore, spectroscopic and microscopic analyses unveiled that S138E and S348E exhibit slower fibrillation kinetics compared to DPF3a WT involving distinct aggregation mechanisms.

Ice-binding proteins are expressed in the cells of some organisms, helping them to survive extremely low temperatures. One of the problems in study of such proteins is the difficulty of isolation and purification. For example, eight cysteine residues in cfAFP from Choristoneura fumiferana (the eastern spruce budworm) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically. In this work we designed a mutant form of ice-binding protein cfAFP, which is much more easy to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and at least does not decrease the freezing temperature.

In Bacillus species, the interaction between the repressor AbrB and the antirepressor AbbA is vital for regulating gene expression. Our study reveals that AbrB binds DNA cooperatively the promoter of the phyC gene through multiple tetramers, forming a complex regulatory mechanism. AbbA disrupts AbrBs DNA binding by competing for its DNA-binding sites, as shown by surface plasmon resonance (SPR) and gel shift assays. Circular dichroism (CD) confirmed that AbbA does not bind directly to the phyC promoter but mimics DNA to interfere with AbrB. Small-angle X-ray scattering (SAXS) data suggest that AbbA resembles a deformed DNA double helix. Our results indicate that AbbA binds to AbrBs DNA-binding sites located with the N-terminal domain causing AbrB displacement. The interaction exhibits negative cooperativity, with both high- and low-affinity binding sites, as evidenced by Scatchard plots and kinetic studies. Our findings suggest that AbbA effectively mimics DNA to displace AbrB, activating transition-state genes. This research enhances our understanding of bacterial gene regulation and provides insights into the complex mechanisms controlling transcription in Bacillus species.

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 M to 200 M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health.

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. Also, it is one of the key cardiac factors crucial for the enhanced derivation of functional and mature myocytes from non-myocyte cells. Here, we report the generation of the recombinant human HAND2 fusion protein from the heterologous system. First, we cloned the full-length human HAND2 gene (only protein-coding sequence) after codon optimization along with the fusion tags (for cell penetration, nuclear translocation, and affinity purification) into the expression vector. We then transformed and expressed it in Escherichia coli (E. coli) strain, BL21(DE3). Next, the effect (in terms of expression) of tagging of fusion tags with this recombinant protein at two different terminals was also investigated. Notably, using affinity chromatography, we established the one-step homogeneous purification of human recombinant HAND2 protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. Furthermore, we show that this purified human protein could transduce the human cells and translocate to its nucleus. Prospectively, the purified recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.