Purification and cloning of nicosulfuron-degrading enzymes from Bacillus subtilis YB1

The nicosulfuron-degrading enzymes from Bacillus subtilis strain YB1 were purified and their genes were cloned. The proteins of bacterial culture filtrate were precipitated with ammonium sulfate or acetone. The extracellular proteins concentrated by acetone were purified from DEAE-Sepharose Fast Flow chromatography. The four protein peaks eluted from DEAE-column were separated and purified by native PAGE. Three components (P1-1, P3-2, P4-3) had nicosulfuron-degrading activity, and component P4-3 degradated 57.5% of this compound. The molecular weights of the components were 33.5, 54.8 and 37.0 kDa, respectively. The amino acid sequences of nicosulfuron-degrading enzymes from B. subtilis YB1 were determined by MALDI-TOF-MS, indicating these enzymes as manganese ABC transporter, vegetative catalase 1 and acetoin dehydrogenase E1, respectively. Using PCR amplification, genes 918, 1428, 1026 bp in size were detected for the enzymes studied.     

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor.     

Computational insight into novel molecular recognition mechanism of different bioactive GAs and the Arabidopsis receptor GID1A

Gibberellin (GA) is an essential plant hormone and plays a significant role during the growth and development of the higher plants. The molecular recognition mode between GA and receptor Arabidopsis thaliana GIBBERELLIN INSENSITIVE DWARF1 A (AtGID1A) was investigated by molecular docking and dynamics simulations to clarify the selective perceived mechanism of different bioactive GA molecules to AtGID1A. The 6-COOH group of GA, especially its β configuration, was found to be an indispensable pharmacophore group for GA recognition and binding to AtGID1A. Not only does a strong salt bridge interaction between the 6β-COOH group of GA and Arg244 of AtGID1A play a very important role in the GA recognition of the receptor, but also an indirect water bridge interaction between the pharmacophore group 6β-COOH of GA and the residue Tyr322 of AtGID1A is essential for the GA binding to the receptor. The site-directed residues mutant modeling study on the receptor-binding pocket confirmed that the mutations of Arg244 and Tyr322 decreased the GA binding activity due to the disappearances of the salt bridge and the hydrogen bond interaction. The 3β-OH group of GA was well known to be necessary for the GA bioactivity due to its forming a unique hydrogen bond with Tyr127 of AtGID1A. In addition, the hydrophobic interaction between GA and AtGID1A was considered a necessary factor to lock the GA active conformation and stabilize the GA-GID1A complex structure. The novel molecular recognition mode will be beneficial in elucidating the GA regulation function on the growth and development of the higher plants.

Structure-Based Computational Study of Two Disease Resistance Gene Homologues (Hm1 and Hm2) in Maize (Zea mays L.) with Implications in Plant-Pathogen Interactions

The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes'' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be −616.989 and −16.9749 kJ mol⁻¹ respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.     

Curcumin specifically binds to the human calcium–calmodulin-dependent protein kinase IV: fluorescence and molecular dynamics simulation studies

Calcium–calmodulin-dependent protein kinase IV (CAMK4) plays significant role in the regulation of calcium-dependent gene expression, and thus, it is involved in varieties of cellular functions such as cell signaling and neuronal survival. On the other hand, curcumin, a naturally occurring yellow bioactive component of turmeric possesses wide spectrum of biological actions, and it is widely used to treat atherosclerosis, diabetes, cancer, and inflammation. It also acts as an antioxidant. Here, we studied the interaction of curcumin with human CAMK4 at pH 7.4 using molecular docking, molecular dynamics (MD) simulations, fluorescence binding, and surface plasmon resonance (SPR) methods. We performed MD simulations for both neutral and anionic forms of CAMK4-curcumin complexes for a reasonably long time (150 ns) to see the overall stability of the protein–ligand complex. Molecular docking studies revealed that the curcumin binds in the large hydrophobic cavity of kinase domain of CAMK4 through several hydrophobic and hydrogen-bonded interactions. Additionally, MD simulations studies contributed in understanding the stability of protein–ligand complex system in aqueous solution and conformational changes in the CAMK4 upon binding of curcumin. A significant increase in the fluorescence intensity at 495 nm was observed (λ_exc = 425 nm), suggesting a strong interaction of curcumin to the CAMK4. A high binding affinity (K_D = 3.7 × 10^?8 ± .03 M) of curcumin for the CAMK4 was measured by SPR further indicating curcumin as a potential ligand for the CAMK4. This study will provide insights into designing a new inspired curcumin derivatives as therapeutic agents against many life-threatening diseases.     

Exploration of the binding of proton pump inhibitors to human P450 2C9 based on docking and molecular dynamics simulation

Human P450 protein CYP2C9 is one of the major drug-metabolizing isomers, contributing to the oxidation of 16% of the drugs currently in clinical use. To examine the interaction mechanisms between CYP2C9 and proton pump inhibitions (PPIs), we used molecular docking and molecular dynamics (MD) simulation methods to investigate the conformations and interactions around the binding sites of PPIs/CYPP2C9. Results from molecular docking and MD simulations demonstrate that nine PPIs adopt two different conformations (extended and U-bend structures) at the binding sites and position themselves far above the heme of 2C9. The presence of PPIs changes the secondary structures and residue flexibilities of 2C9. Interestingly, at the binding sites of all PPI–CYP2C9 complexes except for Lan/CYP2C9, there are hydrogen-bonding networks made of PPIs, water molecules, and some residues of 2C9. Moreover, there are strong hydrophobic interactions at all binding sites for PPIs/2C9, which indicate that electrostatic interactions and hydrophobic interactions appear to be important for stabilizing the binding sites of most PPIs/2C9. However, in the case of Lan/2C9, the hydrophobic interactions are more important than the electrostatic interactions for stabilizing the binding site. In addition, an interesting conformational conversion from extended to U-bend structures was observed for pantoprazole, which is attributed to an H-bond interaction in the binding pocket, an internal π–π stacking interaction, and an internal electrostatic interaction of pantoprazole.     

Inhibitors of catalase-amyloid interactions protect cells from beta-amyloid-induced oxidative stress and toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.     

Cholinesterase inhibitory activities of some flavonoid derivatives and chosen xanthone and their molecular docking studies

Flavonoids are one of the largest classes of plant secondary metabolites and are known to possess a number of significant biological activities for human health. In this study, we examined in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of four flavonoid derivatives - quercetin, rutin, kaempferol 3-O-β-d-galactoside and macluraxanthone. The in vitro results showed that quercetin and macluraxanthone displayed a concentration-dependant inhibition of AChE and BChE. Macluraxanthone showed to be the most potent and specific inhibitor of both the enzymes having the IC₅₀ values of 8.47 and 29.8 μM, respectively. The enzyme kinetic studies revealed that quercetin inhibited both the enzymes in competitive manner, whereas the mode of inhibition of macluraxanthone was non-competitive against AChE and competitive against BChE. The inhibitory profiles of the compounds have been compared with standard AChE inhibitor galanthamine. To get insight of the intermolecular interactions, the molecular docking studies of these two compounds were performed at the active site 3D space of both the enzymes, using ICM-Dock™ module. Docking studies exhibited that macluraxanthone binds much more tightly with both the enzymes than quercetin. The calculated docking and binding energies also supported the in vitro inhibitory profiles (IC₅₀ values). Both the compounds showed several strong hydrogen bonds to several important amino acid residues of both the enzymes. A number of hydrophobic interactions could also explain the potency of the compounds to inhibit AChE and BChE.     

Elucidating the interaction of clofazimine with bovine liver catalase; a comprehensive spectroscopic and molecular docking approach

Nowadays, understanding of interface between protein and drugs has become an active research area of interest. These types of interactions provide structural guidelines in drug design with greater clinical efficacy. Thus, structural changes in catalase induced by clofazimine were monitored by various biophysical techniques including UV‐visible spectrometer, fluorescence spectroscopy, circular dichroism, and dynamic light scattering techniques. Increase in absorption spectra (UV‐visible spectrum) confers the complex formation between drug and protein. Fluorescence quenching with a binding constants of 2.47 × 10⁴ M⁻¹ revealed that clofazimine binds with protein. Using fluorescence resonance energy transfer, the distance (r ) between the protein (donor) and drug (acceptor) was found to be 2.89 nm. Negative Gibbs free energy change (ΔG °) revealed that binding process is spontaneous. In addition, an increase in α‐helicity was observed by far‐UV circular dichroism spectra by adding clofazimine to protein. Dynamic light scattering results indicate that topology of bovine liver catalase was slightly altered in the presence of clofazimine. Hydrophobic interactions are the main forces between clofazimine and catalase interaction as depicted by molecular docking studies. Apart from hydrophobic interactions, some hydrogen bonding was also observed during docking method. The results obtained from the present study may establish abundant in optimizing the properties of ligand‐protein mixtures relevant for numerous formulations.     

Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter

K(ATP) channels are large heteromultimeric complexes containing four subunits from the inwardly rectifying K+ channel family (Kir6.2) and four regulatory sulphonylurea receptor subunits from the ATP-binding cassette (ABC) transporter family (SUR1 and SUR2A/B). The molecular basis for interactions between these two unrelated protein families is poorly understood. Using novel trafficking-based interaction assays, coimmunoprecipitation, and current measurements, we show that the first transmembrane segment (M1) and the N terminus of Kir6.2 are involved in K(ATP) assembly and gating. Additionally, the transmembrane domains, but not the nucleotide-binding domains, of SUR1 are required for interaction with Kir6.2. The identification of specific transmembrane interactions involved in K(ATP) assembly may provide a clue as to how ABC proteins that transport hydrophobic substrates evolved to regulate other membrane proteins.     

VEGF-A regulates sFlt-1 production in trophoblasts through both Flt-1 and KDR receptors

In the present study, we investigated the effect of allyl isothiocyanate (AITC) on liver detoxification signaling pathway in 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis. Mammary tumor was induced by a single dose of DMBA (25 mg/rat) injected subcutaneously near the mammary gland in Sprague–Dawley rats. DMBA-alone-treated rats show an increased synthesis of phase I detoxification enzymes, lipid peroxidative markers, liver marker enzymes, and lipid profiles whereas, depletion of phase II detoxification enzymes and antioxidants in rat liver tissues. Oral administration of AITC restored the levels of biochemical markers in DMBA-treated rats. Furthermore, histopathological results also confirmed that AITC protects DMBA-mediated hepatocellular damage. We also observed that AITC treatment significantly downregulates AhR and upregulates the expression of Nrf2 in DMBA-treated rats. The binding efficacy of AITC with AhR and Nrf2 analysis by molecular docking studies reveals that AITC has strong interaction with AhR and Nrf2 proteins through hydrogen and hydrophobic interactions. Thus, AITC prevents DMBA-induced mammary carcinogenesis via inhibition of phase I and induction of phase II detoxification enzymes by modulating AhR/Nrf2 signaling pathway.     

Novel Interaction of Ornithine Decarboxylase with Sepiapterin Reductase Regulates Neuroblastoma Cell Proliferation

Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH₄), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein–protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC–SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan–Meier analysis (log-rank test, P = 5 × 10^− 4), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.     

Computational investigation of the molecular conformation-dependent binding mode of (<Emphasis Type="Italic">E</Emphasis>)-<Emphasis Type="Italic">β</Emphasis>-farnesene analogs with a heterocycle to aphid odorant-binding proteins

Odorant-binding proteins (OBPs) play an important role as ligand-transfer filters in olfactory recognition in insects. (E)-β-farnesene (EBF) is the main component of the aphid alarm pheromone and could keep aphids away from crops to prevent damage. Computational insight into the molecular binding mode of EBF analogs containing a heterocycle based on the structure of Megoura viciae OBP 3 (MvicOBP3) was obtained by molecular docking and molecular dynamics simulations. The results showed that high affinity EBF analogs substituted with an aromatic ring present a unique binding conformation in the surface cavity of MvicOBP3. A long EBF chain was located inside the cavity and was surrounded by many hydrophobic residues, while the substituted aromatic ring was exposed to the outside due to limitations from the formation of multiple hydrogen bonds. However, the low activity EBF analogs displayed an exactly inverted binding pose, with EBF loaded on the external side of the protein cavity. The affinity of the recently synthesized EBF analogs containing a triazine ring was evaluated in silico based on the binding modes described above and in vitro through fluorescence competitive binding assay reported later. Compound N1 not only showed a similar binding conformation to that of the high affinity analogs but was also found to have a much higher docking score and binding affinity than the other analogs. In addition, the docking score results correlated well with the predicted logP values for these EBF analogs, suggesting highly hydrophobic interactions between the protein and ligand. These studies provide an in silico screening model for the binding affinity of EBF analogs in order to guide their rational design based on aphid OBPs.     

Sequence-specific minor groove binding by bis-benzimidazoles: water molecules in ligand recognition

The binding of two symmetric bis-benzimidazole compounds, 2,2-bis-[4′-(3″-dimethylamino-1″-propyloxy)phenyl]-5,5-bi-1H-benzimidazole and its piperidinpropylphenyl analog, to the minor groove of DNA, have been studied by DNA footprinting, surface plasmon resonance (SPR) methods and molecular dynamics simulations in explicit solvent. The footprinting and SPR methods find that the former compound has enhanced affinity and selectivity for AT sequences in DNA. The molecular modeling studies have suggested that, due to the presence of the oxygen atom in each side chain of the former compound, a water molecule is immobilized and effectively bridges between side chain and DNA base edges via hydrogen bonding interactions. This additional contribution to ligand–DNA interactions would be expected to result in enhanced DNA affinity, as is observed.     

Structure-Based Analysis of the Ligand-Binding Mechanism for DhelOBP21, a C-minus Odorant Binding Protein,from Dastarcus helophoroides (Fairmaire; Coleoptera: Bothrideridae)

Odorant binding proteins (OBPs) transport hydrophobic odor molecules across the sensillar lymph to trigger a neuronal response. Herein, the Minus-C OBP (DhelOBP21) was characterized from Dastarcus helophoroides, the most important natural parasitic enemy insect that targets Monochamus alternatus. Homology modeling and molecular docking were conducted on the interaction between DhelOBP21 and 17 volatile molecules (including volatiles from pine bark, the larva of M. alternatus, and the faeces of the larva). The predicted three-dimensional structure showed only two disulfide bridges and a hydrophobic binding cavity with a short C-terminus. Ligand-binding experiments using N-phenylnaphthylamine (1-NPN) as a fluorescent probe showed that DhelOBP21 exhibited better binding affinities against those ligands with a molecular volume between 100 and 125 ų compared with ligands with a molecular volume between 160 and 185 ų. Molecules that are too big or too small are not conducive for binding. We mutated the amino acid residues of the binding cavity to increase either hydrophobicity or hydrophilia. Ligand-binding experiments and cyber molecular docking assays indicated that hydrophobic interactions are more significant than hydrogen-bonding interactions. Although hydrogen-bond interactions could be predicted for some binding complexes, the hydrophobic interactions had more influence on binding following hydrophobic changes that affected the cavity. The orientation of ligands affects binding by influencing hydrophobic interactions. The binding process is controlled by multiple factors. This study provides a basis to explore the ligand-binding mechanisms of Minus-C OBP.     

In silico modeling and hydrogen peroxide binding study of rice catalase

Homology modeling of the catalase, CatC cloned and sequenced from rice (Oryza sativa L., cv Ratna an Indica cultivar) has been performed based on the crystal structure of the catalase CatF (PDB code 1m7s) by using the software MODELLER. With the aid of molecular mechanics and molecular dynamics methods, the final model is obtained and is further assessed by PROCHECK and VERIFY - 3D graph, which show that the final refined model is reliable. With this model, a flexible docking study with the hydrogen peroxide, the substrate for catalase, is performed and the results indicate that Arg310, Asp343 and Arg346 in catalase are three important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate. These hydrogen-bonding interactions play an important role for the stability of the complex. Our results may be helpful for further experimental investigations.     

An internal docking site stabilizes substrate binding to γ-secretase: Analysis by molecular dynamics simulations

Amyloid precursor protein (APP) is cleaved and processed sequentially by γ-secretase yielding amyloid β (Aβ) peptides of different lengths. Longer Aβ peptides are associated with the formation of neurotoxic plaques related to Alzheimer’s disease. Based on the APP substrate-bound structure of γ-secretase, we investigated the enzyme-substrate interaction using molecular dynamics simulations and generated model structures that represent the sequentially cleaved intermediates during the processing reaction. The simulations indicated an internal docking site providing strong enzyme-substrate packing interaction. In the enzyme-substrate complex, it is located close to the region where the helical conformation of the substrate is interrupted and continues toward the active site in an extended conformation. The internal docking site consists of two non-polar pockets that are preferentially filled by large hydrophobic or aromatic substrate side chains to stabilize binding. Placement of smaller residues such as glycine can trigger a shift in the cleavage pattern during the simulations or results in destabilization of substrate binding. The reduced packing by smaller residues also influences the hydration of the active site and the formation of a catalytically active state. The simulations on processed substrate intermediates and a substrate G33I mutation offer an explanation of the experimentally observed relative increase of short Aβ fragment production for this mutation. In addition, studies on a substrate K28A mutation indicate that the internal docking site opposes the tendency of substrate dissociation due to a hydrophobic mismatch at the membrane boundary caused by K28 during processing and substrate movement toward the enzyme active site. The proposed internal docking site could also be useful for the specific design of new γ-secretase modulators.     

Insight into the binding mode of a novel LSD1 inhibitor by molecular docking and molecular dynamics simulations

Abstract

Lysine-specific demethylase (LSD1) is an important enzyme for histone lysine methylation. Downregulated LSD1 expression has been linked to cancer proliferation, migration and invasion, indicating that it is an important target for anti-cancer medication. In the present study, the binding modes of a recent reported new series of LSD1 inhibitor were analyzed by using molecular docking and molecular dynamics simulations. A binding mode of these inhibitors was proposed based on the results. According to this binding mode, Thr628 can form two important hydrogen bonds with these inhibitors. Moreover, if the inhibitors can form an additional hydrogen bond with hydroxyl group of Ser289, the potency of the inhibitor can be greatly improved, such as the best inhibitor (compound 12d) in this series. Hydrophobic interactions between the inhibitors and LSD1 are also key contributor here, such as the interaction between the hydrophobic groups (benzene rings) of the inhibitors and the hydrophobic residues of LSD1 (including Val288, Val317, Val811, Ala814, Leu659, Trp751 and Tyr761). Based on the results and analysis, it may provide some useful information for future novel LSD1 inhibitor design.     

Investigation on the interaction of catalase with sodium lauryl sulfonate and the underlying mechanisms

As a classic type of anionic surfactants, sodium lauryl sulfonate (SLS) might change the structure and function of antioxidant enzyme catalase (CAT) through their direct interactions. However, the underlying molecular mechanism is still unknown. This study investigated the direct interaction of SLS with CAT molecule and the underlying mechanisms using multi‐spectroscopic methods, isothermal titration calorimetry, and molecular docking studies. No obvious effects were observed on CAT structure and activity under low SLS concentration exposure. The particle size of CAT molecule decreased and CAT activity was slightly inhibited under high SLS concentration exposure. SLS prefers to bind to the interface of CAT mainly via van der Waals’ forces and hydrogen bonds. Subsequently, SLS interacts with the amino acid residues around the heme groups of CAT via hydrophobic interactions and might inhibit CAT activity.