Corrigendum: The mycoremediation potential of the armillarioids: a comparative genomics analysis.

[This corrects the article DOI: 10.3389/fbioe.2023.1189640.].

Modeling the Effect on a Novel Fungal Peptaibol Placed in an All-Atom Bacterial Membrane Mimicking System via Accelerated Molecular Dynamics Simulations.

We previously reported on a novel peptaibol, named Tripleurin XIIc (TPN), an 18-residue long sequence produced by the fungus Trichoderma pleuroti. We elucidated its 3D structure via classical and accelerated molecular dynamics simulation (aMD) methods and reported the folding dynamics of TPN in water and chloroform solvents. Peptaibols, in general, are insoluble in water, as they are amphipathic and may prefer hydrophobic environments like transmembrane regions. In this study, we attempted to use aMD simulations to model an all-atom bacterial membrane system while placing a TPN molecule in its vicinity. The results highlighted that TPN was able to introduce some disorder into the membrane and caused lipid clustering. It could also enter the transmembrane region from the water-bilayer interface. The structural dynamics of TPN in the transmembrane region revealed a single energetically stable conformation similar to the one obtained from water and chloroform solvent simulations reported by us previously. However, this linear structure was found to be at the local energy minimum (stable) in water but at a metastable intermediate state (higher energy) in chloroform. Therefore, it could be said that the water solvent can be successfully used for folding simulations of peptaibols.

The mycoremediation potential of the armillarioids: a comparative genomics analysis.

Genes involved in mycoremediation were identified by comparative genomics analysis in 10 armillarioid species and selected groups of white-rot Basidiomycota (14) and soft-rot Ascomycota (12) species to confine the distinctive bioremediation capabilities of the armillarioids. The genomes were explored using phylogenetic principal component analysis (pPCA), searching for genes already documented in a biocatalysis/biodegradation database. The results underlined a distinct, increased potential of aromatics-degrading genes/enzymes in armillarioids, with particular emphasis on a high copy number and diverse spectrum of benzoate 4-monooxygenase [EC:1.14.14.92] homologs. In addition, other enzymes involved in the degradation of various monocyclic aromatics were more abundant in the armillarioids than in the other white-rot basidiomycetes, and enzymes involved in the degradation of polycyclic aromatic hydrocarbons (PAHs) were more prevailing in armillarioids and other white-rot species than in soft-rot Ascomycetes. Transcriptome profiling of A. ostoyae and A. borealis isolates confirmed that several genes involved in the degradation of benzoates and other monocyclic aromatics were distinctively expressed in the wood-invading fungal mycelia. Data were consistent with armillarioid species offering a more powerful potential in degrading aromatics. Our results provide a reliable, practical solution for screening the likely fungal candidates for their full biodegradation potential, applicability, and possible specialization based on their genomics data.

Unraveling the Mechanism of Action of Myricetin in the Inhibition of hUba1∼Ubiquitin Thioester Bond Formation via In Silico Molecular Modeling Techniques.

Ubiquitination is a crucial type of protein modification which helps to control substrate degradation and maintain cell homeostasis. Recent studies suggest that ubiquitination and deubiquitination are involved in regulating metabolic reprogramming in cancer cells and maintaining cancer stem cells. Uba1, a crucial protein in the ubiquitination cascade, can be targeted to develop effective inhibitors for cancer treatment. In previous work, we showed that myricetin (Myr) acts as a potential human Uba1 (hUba1) inhibitor. In this study, we have utilized computational modeling techniques to attempt to illustrate the mechanism of action of Myr. Through extra-precision docking, we confirmed that Myr binds to the adenosine triphosphate (ATP)-binding site of hUba1 (referred to as hotspot 1) with the highest binding affinity. The dynamics of this interaction revealed that hUba1 undergoes a conformational shift from open to closed upon binding of Myr. Myr also migrates outward to interact with the crossover loop simultaneously as the rotational shift of the ubiquitin fold domain (UFD) takes place, thereby blocking access to the ubiquitin binding interface of hUba1 and the crossover loop. The outward migration also explains the reversible nature of Myr binding to hUba1 in previous experiments. We hypothesize that Myr acts as an inhibitor of Uba1∼Ub thioester bond formation by causing a large domain shift toward a closed conformation. Few other analogues of Myr containing the same flavone skeleton showed promising docking scores against hUba1 and could be considered for further validation. We propose that Myr and some of its analogues reported in this study may be promising candidates for developing effective Uba1 inhibitors for cancer treatment.

Structure-activity correlations for peptaibols obtained from clade Longibrachiatum of Trichoderma: A combined experimental and computational approach.

Integrated disease management and plant protection have been discussed with much fervor in the past decade due to the rising environmental concerns of using industrially produced pesticides. Members of the genus Trichoderma are a subject of considerable research today due to their several properties as biocontrol agents. In our study, the peptaibol production of Trichoderma longibrachiatum SZMC 1775, T. longibrachiatum f. bissettii SZMC 12546, T. reesei SZMC 22616, T. reesei SZMC 22614, T. saturnisporum SZMC 22606 and T. effusum SZMC 22611 were investigated to elucidate structure-activity relationships (SARs) between the properties of peptaibols and their 3D structures. The effects of peptaibol mixtures obtained from every Trichoderma strain were examined against nine commonly known bacteria. The lowest minimum inhibitory concentrations (MIC, mg ml-1) were exerted by T. longibrachiatum f. bissettii SZMC 12546 against Gram-positive bacteria, which was also able to inhibit the plant pathogenic Gram-negative Rhizobium radiobacter. Accelerated molecular dynamics (aMD) simulations were performed in aqueous solvent to explore the folding dynamics of 12 selected peptaibol sequences. The most characteristic difference between the peptaibols from group A and B relies in the 'Gly-Leu-Aib-Pro' and 'Gly-Aib-Aib-Pro' motifs ('Aib' stands for α-aminoisobutyric acid), which imparted a significant effect on the folding dynamics in water and might be correlated with their expressed bioactivity. In our aMD simulation experiments, Group A peptaibols showed more restricted folding dynamics with well-folded helical conformations as the most stable representative structures. This structural stability and dynamics may contribute to their bioactivity against the selected bacterial species.

Correction: Tyagi, C., et al. Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem. International Journal of Molecular Sciences, 2019, 20, 4268.

The authors wish to make the following corrections to this paper [...].

Mode of inhibitory binding of epigallocatechin gallate to the ubiquitin-activating enzyme Uba1 via accelerated molecular dynamics.

The green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) and some of its analogs potently inhibit the ubiquitin-activating enzyme Uba1. In an effort to understand the possible molecular basis of inhibitory activity of EGCG, we conducted a molecular docking and molecular dynamics simulation study. We found that EGCG and its two selected analogs, (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin (EGC), bind favorably at two likely hot spots for small-molecule ligand binding on human Uba1. The compounds bind with energetics that mirror their experimental potency for inhibition of Uba1∼ubiquitin thioester formation. The binding of EGCG, ECG, and EGC at one of the hot spots, in particular, recapitulates the rank order of potency determined experimentally and suggests a possible mechanism for inhibition. A hinge-like conformational change of the second catalytic cysteine domain and the opposing ubiquitin-fold domain observed during accelerated molecular dynamics simulations of the EGCG-bound Uba1 complex that results in disruption of the ubiquitin-binding interfaces could explain the compounds' inhibitory activity. These results shed light on the possible molecular mechanism of EGCG and related catechins in the inhibition of Uba1.

Corrigendum: Structural Diversity and Bioactivities of Peptaibol Compounds From the Longibrachiatum Clade of the Filamentous Fungal Genus Trichoderma.

[This corrects the article DOI: 10.3389/fmicb.2019.01434.].

Analysis of mutations of defensin protein using accelerated molecular dynamics simulations.

Plant defensins possess diverse biological functions that include antifungal and antibacterial activities and α-amylase and trypsin inhibitory properties. Two mutations, G9R and V39R, were confirmed to increase the antifungal activity of Raphanus sativus antifungal protein 2 (RsAFP2). Accelerated Molecular Dynamics (aMD) were carried out to examine the conformational changes present in these RsAFP2 mutants, and its two closest homologs compared to the wild-type protein. Specifically, the root mean square fluctuation values for the eight cysteine amino acids involved in the four disulfide bonds were low in the V39R mutant compared to the wild-type. Additionally, analysis of the free energy change revealed that G9R and V39R mutations exert a neutral and stabilizing effect on RsAFP2 conformation, and this is supported by the observed lower total energy of mutants compared to the wild-type, suggesting that enhanced stability of the mutants. However, MD simulations to a longer time scale would aid in capturing more conformational state of the wild-type and mutants defensin protein. Furthermore, the aMD simulations on fungal mimic membranes with RsAFP2 and its mutants and homologs showed that the mutant proteins caused higher deformation and water diffusion than the native RsAFP2, especially the V39R mutant. The mutant variants seem to interact by specifically targeting the POPC and POPI lipids amongst others. This work highlights the stabilizing effect of mutations at the 9th and 39th positions of RsAFP2 and their increased membrane deformation activity.

Molecular Modeling of Chemosensory Protein 3 from Spodoptera litura and Its Binding Property with Plant Defensive Metabolites.

Chemosensory perception in insects involves a broad set of chemosensory proteins (CSPs) that identify the bouquet of chemical compounds present in the external environment and regulate specific behaviors. The current study is focused on the Spodoptera litura (Fabricius) chemosensory-related protein, SlitCSP3, a midgut-expressed CSP, which demonstrates differential gene expression upon different diet intake. There is an intriguing possibility that SlitCSP3 can perceive food-derived chemical signals and modulate insect feeding behavior. We predicted the three-dimensional structure of SlitCSP3 and subsequently performed an accelerated molecular dynamics (aMD) simulation of the best-modeled structure. SlitCSP3 structure has six α-helices arranged as a prism and a hydrophobic binding pocket predominated by leucine and isoleucine. We analyzed the interaction of selected host plant metabolites with the modeled structure of SlitCSP3. Out of two predicted binding pockets in SlitCSP3, the plant-derived defensive metabolites 2-b-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA), 6-Methoxy-2-benzoxazolinone (MBOA), and nicotine were found to interact preferably to the hydrophobic site 1, compared to site 2. The current study provides the potential role of CSPs in recognizing food-derived chemical signals, host-plant specialization, and adaptation to the varied ecosystem. Our work opens new perspectives in designing novel pest-management strategies. It can be further used in the development of CSP-based advanced biosensors.

Accelerated Molecular Dynamics Applied to the Peptaibol Folding Problem.

The use of enhanced sampling molecular dynamics simulations to facilitate the folding of proteins is a relatively new approach which has quickly gained momentum in recent years. Accelerated molecular dynamics (aMD) can elucidate the dynamic path from the unfolded state to the near-native state, "flattened" by introducing a non-negative boost to the potential. Alamethicin F30/3 (Alm F30/3), chosen in this study, belongs to the class of peptaibols that are 7-20 residue long, non-ribosomally synthesized, amphipathic molecules that show interesting membrane perturbing activity. The recent studies undertaken on the Alm molecules and their transmembrane channels have been reviewed. Three consecutive simulations of ~900 ns each were carried out where N-terminal folding could be observed within the first 100 ns, while C-terminal folding could only be achieved almost after 800 ns. It took ~1 µs to attain the near-native conformation with stronger potential boost which may take several µs worth of classical MD to produce the same results. The Alm F30/3 hexamer channel was also simulated in an E. coli mimicking membrane under an external electric field that correlates with previous experiments. It can be concluded that aMD simulation techniques are suited to elucidate peptaibol structures and to understand their folding dynamics.

Voltammetric measurements of neurotransmitter-acetylcholine through metallic nanoparticles embedded 2-D material.

The most generally spread neurotransmitter acetylcholine (Ach) is used as a chemical messenger assisting in conveying signals transversely through the nerve synapse. Herein, two enzymes acetylcholinesterase and choline oxidase were covalently immobilized over the gold nanoparticles (AuNPs) embedded graphene oxide (GO; 2D carbon material) nanocomposite modified ITO coated glass plate. The synergetic and unique properties of AuNPs and GO present in nanocomposite are used to detect the ultra-small concentration of analyte, Ach. The prepared nanocomposites were characterized using different techniques i.e. TEM, XRD, SEM, FTIR, UV-Vis and Raman Spectroscopy. All the electrochemical measurements were performed using 3 electrodes integrated electrochemical system by introducing Ach through varying its concentration from 100 pM to 1000 nM. Cyclic voltammetry curves for different concentrations of Ach indicate the facile charge transfer process over the working electrode. Square wave voltammetry curves indicate the good sensing measurements of the modified electrode at the fixed potential. The limit of detection was found to be as low as 100 pM. In addition to these, selectivity of the electrode towards Ach molecule was confirmed by measuring the response towards other interfering agents. Besides this, the present nano interface is capable of detecting Ach in biological fluid such as serum.

Structural Diversity and Bioactivities of Peptaibol Compounds From the Longibrachiatum Clade of the Filamentous Fungal Genus Trichoderma.

This study examined the structural diversity and bioactivity of peptaibol compounds produced by species from the phylogenetically separated Longibrachiatum Clade of the filamentous fungal genus Trichoderma, which contains several biotechnologically, agriculturally and clinically important species. HPLC-ESI-MS investigations of crude extracts from 17 species of the Longibrachiatum Clade (T. aethiopicum, T. andinense, T. capillare, T. citrinoviride, T. effusum, T. flagellatum, T. ghanense, T. konilangbra, T. longibrachiatum, T. novae-zelandiae, T. pinnatum, T. parareesei, T. pseudokoningii, T. reesei, T. saturnisporum, T. sinensis, and T. orientale) revealed several new and recurrent 20-residue peptaibols related to trichobrachins, paracelsins, suzukacillins, saturnisporins, trichoaureocins, trichocellins, longibrachins, hyporientalins, trichokonins, trilongins, metanicins, trichosporins, gliodeliquescins, alamethicins and hypophellins, as well as eight 19-residue sequences from a new subfamily of peptaibols named brevicelsins. Non-ribosomal peptide synthetase genes were mined from the available genome sequences of the Longibrachiatum Clade. Their annotation and product prediction were performed in silico and revealed full agreement in 11 out of 20 positions regarding the amino acids predicted based on the signature sequences and the detected amino acids incorporated. Molecular dynamics simulations were performed for structural characterization of four selected peptaibol sequences: paracelsins B, H and their 19-residue counterparts brevicelsins I and IV. Loss of position R6 in brevicelsins resulted in smaller helical structures with higher atomic fluctuation for every residue than the structures formed by paracelsins. We observed the formation of highly bent, almost hairpin-like, helical structures throughout the trajectory, along with linear conformation. Bioactivity tests were performed on the purified peptaibol extract of T. reesei on clinically and phytopathologically important filamentous fungi, mammalian cells, and Arabidopsis thaliana seedlings. Porcine kidney cells and boar spermatozoa proved to be sensitive to the purified peptaibol extract. Peptaibol concentrations ≥0.3 mg ml-1 deterred the growth of A. thaliana. However, negative effects to plants were not detected at concentrations below 0.1 mg ml-1, which could still inhibit plant pathogenic filamentous fungi, suggesting that those peptaibols reported here may have applications for plant protection.

Tripleurin XIIc: Peptide Folding Dynamics in Aqueous and Hydrophobic Environment Mimic Using Accelerated Molecular Dynamics.

Peptaibols are a special class of fungal peptides with an acetylated N-terminus and a C-terminal 1,2-amino alcohol along with non-standard amino acid residues. New peptaibols named tripleurins were recently identified from a strain of the filamentous fungal species Trichoderma pleuroti, which is known to cause green mould disease on cultivated oyster mushrooms. To understand the mode of action of these peptaibols, the three-dimensional structure of tripleurin (TPN) XIIc, an 18-mer peptide, was elucidated using an enhanced sampling method, accelerated MD, in water and chloroform solvents. Non-standard residues were parameterized by the Restrained Electrostatic Potential (RESP) charge fitting method. The dihedral distribution indicated towards a right-handed helical formation for TPN XIIc in both solvents. Dihedral angle based principal component analysis revealed a propensity for a slightly bent, helical folded conformation in water solvent, while two distinct conformations were revealed in chloroform: One that folds into highly bent helical structure that resembles a beta-hairpin and another with an almost straight peptide backbone appearing as a rare energy barrier crossing event. The hinge-like movement of the terminals was also observed and is speculated to be functionally relevant. The convergence and efficient sampling is addressed using Cartesian PCA and Kullback-Leibler divergence methods.

New 19-Residue Peptaibols from Trichoderma Clade Viride.

Trichoderma koningiopsis and T. gamsii belong to clade Viride of Trichoderma, the largest and most diverse group of this genus. They produce a wide range of bioactive secondary metabolites, including peptaibols with antibacterial, antifungal, and antiviral properties. The unusual amino acid residues of peptaibols, i.e., α-aminoisobutyric acid (Aib), isovaline (Iva), and the C-terminal 1,2-amino alcohol make them unique among peptides. In this study, the peptaibiomes of T. koningiopsis and T. gamsii were investigated by HPLC-ESI-MS. The examined strains appeared to produce 19-residue peptaibols, most of which are unknown from literature, but their amino acid sequences are similar to those of trikoningins, tricholongins, trichostrigocins, trichorzianins, and trichorzins. A new group of peptaibols detected in T. koningiopsis are described here under the name "Koningiopsin". Trikoningin KA V, the closest peptaibol compound to the peptaibols produced by these two strains, was selected for structural investigation by short MD simulation, which revealed that many residues show high preference for left handed helix formation. The bioactivity of the peptaibol mixtures produced by T. koningiopsis and T. gamsii was tested on agar plates against bacteria, yeasts, and filamentous fungi. The results revealed characteristic differences in bioactivities towards the different groups of target microorganisms, which can be explained with the differences in their cell wall structures.

In silico prediction of active site and in vitro DNase and RNase activities of Helicoverpa-inducible pathogenesis related-4 protein from Cicer arietinum.

Plants are endowed with an innate immune system, which enables them to protect themselves from pest and pathogen. The participation of pathogenesis-related (PR) proteins is one of the most crucial events of inducible plant defense response. Herein, we report the characterization of CaHaPR-4, a Helicoverpa-inducible class II PR-4 protein from chickpea. Bioinformatic analysis of CaHaPR-4 protein indicated the presence of a signal peptide, barwin domain but it lacks the chitin-binding site/hevein domain. The recombinant CaHaPR-4 is bestowed with RNase and bivalent ion-dependent DNase activity. Further, the RNA and DNA binding sites were identified and confirmed by analyzing interactions between mutated CaHaPR-4 with the altered active site and ribonuclease inhibitor, 5'ADP and DNase inhibitor, 2­nitro­5­thiocyanobenzoic acid (NTCB) using 3D modeling and docking studies. Moreover, CaHaPR-4 shows antifungal activity as well as growth inhibiting properties against neonatal podborer larvae. To the best of our knowledge, this is the first report of a PR-4 showing RNase, DNase, antifungal and most importantly insect growth inhibiting properties against Helicoverpa armigera simultaneously.

Dynamics of fluoroquinolones induced resistance in DNA gyrase of Mycobacterium tuberculosis.

DNA gyrase is a validated target of fluoroquinolones which are key components of multidrug resistance tuberculosis (TB) treatment. Most frequent occurring mutations associated with high level of resistance to fluoroquinolone in clinical isolates of TB patients are A90V, D94G, and A90V-D94G (double mutant [DM]), present in the larger subunit of DNA Gyrase. In order to explicate the molecular mechanism of drug resistance corresponding to these mutations, molecular dynamics (MD) and mechanics approach was applied. Structure-based molecular docking of complex comprised of DNA bound with Gyrase A (large subunit) and Gyrase C (small subunit) with moxifloxacin (MFX) revealed high binding affinity to wild type with considerably high Glide XP docking score of -7.88 kcal/mol. MFX affinity decreases toward single mutants and was minimum toward the DM with a docking score of -3.82 kcal/mol. Docking studies were also performed against 8-Methyl-moxifloxacin which exhibited higher binding affinity against wild and mutants DNA gyrase when compared to MFX. Molecular Mechanics/Generalized Born Surface Area method predicted the binding free energy of the wild, A90V, D94G, and DM complexes to be -55.81, -25.87, -20.45, and -12.29 kcal/mol, respectively. These complexes were further subjected to 30 ns long MD simulations to examine significant interactions and conformational flexibilities in terms of root mean square deviation, root mean square fluctuation, and strength of hydrogen bond formed. This comparative drug interaction analysis provides systematic insights into the mechanism behind drug resistance and also paves way toward identifying potent lead compounds that could combat drug resistance of DNA gyrase due to mutations.

Helicoverpa-inducible Thioredoxin h from Cicer arietinum: structural modeling and potential targets.

Thioredoxins are small and universal proteins, which are involved in the cell redox regulation. In plants, they participate in a broad range of biochemical processes like self-incompatibility, seed germination, pathogen & pest defense and oxidative stress tolerance. The h-type of thioredoxin (Trx-h) protein represents the largest Trx family. Herein, we characterized the Helicoverpa - inducible Trx h from an important legume, Cicer arietinum, CaHaTrx-h, 'CGFS' type Trxs, which encodes for a 113 amino acids long protein and possess characteristic motifs "FLKVDVDE" and "VVDFTASWCGPCRFIAPIL" and 73% sequence identity with AtTrx-h. Homology modeling and simulation of the target showed that the extended ß-sheet regions remain stable during the simulation while the helical regions fluctuate between alpha and 3-10 helical forms and highlights the flexibility of helix2-helix3 and terminal regions probably to accommodate an approaching protein target and facilitate their interaction. During the simulation, the structure exists in five energy minima clusters with biggest cluster size belonging to 20-25 ns time frames. PR-5 and Mannitol Dehydrogenase were nominated as potential targets and share close interaction with CaHaTrx-h via disulfide bond reduction. The study is an effort in the direction of understanding stress-related mechanisms in crop plants to overcome losses in agricultural yield.

Natural polyphenolic inhibitors against the antiapoptotic BCL-2.

The apoptotic mechanism is regulated by the BCL-2 family of proteins, such as BCL-2 or Bcl-xL, which block apoptosis while Bad, Bak, Bax, Bid, Bim or Hrk induce apoptosis. The overexpression of BCL-2 was found to be related to the progression of cancer and also providing resistance towards chemotherapeutic treatments. In the present study, we found that all polyphenols (apigenin, fisetin, galangin and luteolin) bind to the hydrophobic groove of BCL-2 and the interaction is stable throughout MD simulation run. Luteolin was found to bind with highest negative binding energy and thus, claimed highest potency towards BCL-2 inhibition followed by fisetin. The hydrophobic interactions were found to be critical for stable complex formation as revealed by the vdW energy and ligplot analysis. Finally, on the basis of data obtained during the study, it can be concluded that these polyphenols have the potential to be used as lead molecules for BCL-2 inhibition.

Diversity Profile and Dynamics of Peptaibols Produced by Green Mould Trichoderma Species in Interactions with Their Hosts Agaricus bisporus and Pleurotus ostreatus.

Certain Trichoderma species are causing serious losses in mushroom production worldwide. Trichoderma aggressivum and Trichoderma pleuroti are among the major causal agents of the green mould diseases affecting Agaricus bisporus and Pleurotus ostreatus, respectively. The genus Trichoderma is well-known for the production of bioactive secondary metabolites, including peptaibols, which are short, linear peptides containing unusual amino acid residues and being synthesised via non-ribosomal peptide synthetases (NRPSs). The aim of this study was to get more insight into the peptaibol production of T. aggressivum and T. pleuroti. HPLC/MS-based methods revealed the production of peptaibols closely related to hypomurocins B by T. aggressivum, while tripleurins representing a new group of 18-residue peptaibols were identified in T. pleuroti. Putative NRPS genes enabling the biosynthesis of the detected peptaibols could be found in the genomes of both Trichoderma species. In vitro experiments revealed that peptaibols are potential growth inhibitors of mushroom mycelia, and that the host mushrooms may have an influence on the peptaibol profiles of green mould agents.