Main Binding Site Research Articles

Silicon relieves aluminum‐induced inhibition of cell elongation in rice root apex by reducing the deposition of aluminum in the cell wall

Silicon (Si) is known to alleviate aluminum (Al) toxicity in a number of plant species; however, the mechanisms of Si-triggered Al detoxification have not been elucidated, especially in rice (Oryza sativa). We investigated the interactions between Si and Al in root and the role of cell wall polysaccharides in rice resistance to Al. Rice seedlings were grown in 0.5 mM CaCl2 solution with or without 50 µM Al and 1.0 mM H4SiO4 for 24 h. The cell expansion and oxidative injury of root were measured by histochemical analyses; Al accumulation and distribution in root apices were determined by inductively coupled plasma mass spectrometry (ICP-MS); the degree of pectin methylesterification was analyzed by immunofluorescence, and the expression of genes (e.g. OsPME) related to Al resistance was measured by quantitative real-time PCR. Si relieved the inhibition of cell elongation and oxidative injury caused by Al and significantly reduced Al content (by about 41 %) in cell wall (CW). Meanwhile, Si inhibited pectin methylesterase (PME) activity and its gene expression, thereby increasing the degree of pectin methylesterification, and reduced the content of hemicellulose, which was the main binding site for Al in CW, under Al stress. Furthermore, Si decreased the non-exchangeable Al fraction of CW in root, but did not change the cation exchange capacity of CW. Si reduces the deposition of Al in the cell wall of rice root apex by decreasing both the degree of pectin demethylesterification and the content of hemicellulose, thereby alleviating Al stress.

Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry.

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.

Isolation of a novel calcium-binding peptide from phosvitin hydrolysates and the study of its calcium chelation mechanism

A new peptide with strong calcium binding capacity was isolated from phosvitin hydrolysates. Taking calcium chelating rate as an indicator, phosvitin hydrolysates were separated gradually by anion-exchange chromatography, gel filtration chromatography and reversed-phase high performance liquid chromatography. A peptide with a molecular weight of 1106.44402 Da was identified by liquid chromatography-electrospray/mass spectrometry (LC-ESI/MS), and its amino acid sequence was DEEENDQVK, the calcium binding capacity reached 151.10 ± 3.57 mg/g. Its chelating mechanism was investigated. Results showed that, the β-sheet structure of peptide increased after adding calcium ion, and the main binding sites were carboxyl oxygen atom and amino nitrogen atom. In vitro simulated digestion experiments showed that, the solubility and dialysis rate of calcium in peptide-calcium chelate were higher than those in CaCO3 and D-calcium gluconate. This finding would promote the development of calcium supplements from food resources.

Inhibitory capacity of chloroquine against SARS-COV-2 by effective binding with angiotensin converting enzyme-2 receptor: An insight from molecular docking and MD-simulation studies

The main binding site for SARS-COV-2 spike protein in human body is human Angiotensin converting enzyme 2 (ACE2) protein receptor. Herein we present the effect of chloroquine (CLQ) on human ACE2 receptor. Molecular docking studies showed that chloroquine have a docking score is quite high compare to other well known drugs. Furthermore, molecular dynamics (MD) studies with CLQ docked ACE2 results in large fluctuations on RMSD up to 2.3 ns, indicating conformational and rotational changes due to the presence of drug molecule in the ACE2 moiety. Analysis of results showed that CLQ can effect the conformation of human ACE2 receptor. We believed that this work will help researchers to understand better the effect of CLQ on ACE2.

Bis-indolylation of aldehydes and ketones using silica-supported FeCl3: molecular docking studies of bisindoles by targeting SARS-CoV-2 main protease binding sites.

We report herein an operationally simple, efficient and versatile procedure for the synthesis of bis-indolylmethanes via the reaction of indoles with aldehydes or ketones in the presence of silica-supported ferric chloride under grindstone conditions. The prepared supported catalyst was characterized by SEM and EDX spectroscopy. The present protocol has several advantages such as shorter reaction time, high yield, avoidance of using harmful organic solvents during the reaction and tolerance of a wide range of functional groups. Molecular docking studies targeted toward the binding site of SARS-CoV-2 main protease (3CLpro or Mpro) enzymes were investigated with the synthesized bis-indoles. Our study revealed that some of the synthesized compounds have potentiality to inhibit the SARS-CoV-2 Mpro enzyme by interacting with key amino acid residues of the active sites via hydrophobic as well as hydrogen bonding interactions.

Substrate Binding in the Mitochondrial ADP/ATP Carrier is a Step-Wise Process Guiding the Structural Changes in the Transport Cycle

SummaryMitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes. Structures of the inhibited cytoplasmic- and matrix-open states have confirmed an alternating access transport mechanism, but the molecular nature of substrate binding remains unresolved. Here, we evaluate the role of all solvent-exposed residues of the translocation pathway in the process of substrate binding. We identify the main binding site, comprising three positively charged and a set of aliphatic and aromatic residues, which bind ADP and ATP in both states. Additionally, there are two pairs of arginine-asparagine residues on opposite sides of this site that are involved in substrate binding in a state-dependent manner. Thus, the substrates are put in a series of binding poses, inducing the conformational changes of the carrier that lead to their translocation. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.

Complementary chain competition and fluorescence quenching detection of Deoxynivalenol and analytical applications using a novel aptamer

ABSTRACT Deoxynivalenol (DON) is a mycotoxin commonly found in cereals and animal feeds, which also is a carcinogen easily soluble in water. So it has drawn considerable interest in food safety. In this study, a specific aptamer named DON-A16 was developed from an immobilized ssDNA library by affinity chromatography column-based the SELEX. The complementary chain competition method for DON detection was established, and its limit of detection (LOD) was 488 ng/mL. Graphene oxide-based fluorescence quenching was developed using DON-A16 with fluorescein (FAM) label; its LOD was 200 ng/mL, and the detection time was 3 h. The recovery rate of fluorescence quenching was also detected in different samples (oat, wheat flour, corn), which showed the commercial DON ELISA kit led to an increase in repeatability, satisfactory recovery and consistency. The three-dimensional structure and the main binding site of of DON-A16 was ultimately obtained by molecular docking simulation.

Luteolin decreases the pathogenicity of Aeromonas hydrophila via inhibiting the activity of aerolysin

ABSTRACT Aeromonas hydrophila (A. hydrophila) can cause a number of diseases in both human and animals. A. hydrophila-related infections in aquaculture cause severe economic losses every year throughout the world. The emergence of antibiotic resistance that is due to the abuse of antibiotics has limited the application of antibiotics. Thus, novel approaches are needed to combat with treatment failure of antibiotics caused by resistant bacterial strains. Aerolysin plays a critical role in the pathogenesis of A. hydrophila and has been considered as a novel target for developing drugs based on anti-virulence strategies. Here, we reported that luteolin, a natural product with no anti-A. hydrophila activity, could reduce aerolysin-induced hemolysis by inhibiting aerolysin activity. The binding mode was simulated by molecular docking and dynamics simulation. Then the main binding sites were confirmed by fluorescence quenching assays. We found that luteolin could hindered the formation of functional heptamer of aerolysin according to the results of the oligomerization assay. Moreover, luteolin could protect A549 cells from aerolysin mediated cell death and increase the survival rate of A. hydrophila-infected channel catfish. These findings suggest a novel approach to developing drugs fighting against A. hydrophila, and luteolin can be a promising drug candidate for treatment of A. hydrophila-associated infections.

Cu2+, Ca2+, and methionine oxidation expose the hydrophobic α-synuclein NAC domain

α-Synuclein is an intrinsically disordered protein whose aggregation is related to Parkinson's disease and other neurodegenerative disorders. Metal cations are one of the main factors affecting the propensity of α-synuclein to aggregate, either by directly binding to it or by catalyzing the production of reactive oxygen species that oxidize it. His50, Asp121 and several additional C-terminal α-synuclein residues are binding sites for numerous metal cations, while methionine sulfoxidation occurs readily on this protein under oxidative stress conditions. Molecular dynamics simulations are an excellent tool to obtain a microscopic picture of how metal binding or methionine sulfoxidation alter the conformational preferences of α-synuclein and, hence, its aggregation propensity. In this work, we report the first coarse-grained molecular dynamics study comparing the conformational ensembles of the native protein, the protein bound to either Cu2+ or Ca2+ at its main binding sites, and the methionine-sulfoxidized protein. Our results suggest that these events alter the transient α-synuclein intramolecular contacts, inducing a greater solvent exposure of its hydrophobic, aggregation-prone NAC domain, in full agreement with a recent experimental study on Ca2+ binding. Moreover, metal-binding residues directly participate in the long-range contacts that shield this domain and regulate α-synuclein aggregation. These results provide a molecular-level rationalization of the enhanced fibrillation experimentally observed in the presence of Cu2+ or Ca2+ and the oligomerization induced by methionine sulfoxidation.

The relationship between fluoride accumulation in tea plant and changes in leaf cell wall structure and composition under different fluoride conditions

Tea plant is capable of hyper-accumulating fluoride (F) in leaves, suggesting drinking tea may cause excessive F intake in our body and threaten the health. This study investigated the changes in the structure, composition, and F content in the leaf cell wall of the tea (Camellia sinensis) under different F conditions to demonstrate the role of cell wall in F enrichment in tea plants. The cell wall was shown as the main part for F accumulation (67%-92%), with most of F distributed in the pectin fraction (56%-71%). With increasing F concentration, a significant increase (p<0.05) was observed in the F content of cell wall and its components, the level of cell wall metal ions (i.e. Cu, Mg, Zn, Al, Ca, Ba, Mn), as well as the content of total cell wall materials, cellulose, and pectin. Meanwhile, the level of Cu, Mg, Zn, pectin, and cellulose was significantly positively correlated with the F content in the leaf cell wall. F addition was shown to increase the fluorescence intensity of LM19 and 2F4 antibody-labeled low-methylesterified homogalacturonans (HGs), while decrease LM20-labeled high-methylesterified HGs, coupled with an increase in the activity and gene expression of pectin methyl esterases (PMEs) in tea leaves. All these results suggest that F addition can increase pectin content and demethylesterification, leading to increased absorption of metal cations and chelation of F in the cell wall through the action of metal ions.

Identification, taste characterization and molecular docking study of novel umami peptides from the Chinese anchovy sauce.

Fish sauce has a subtle flavor with prominent umami and salty taste, and is accompanied by a certain sweetness and bitterness. In order to identify a wider range of umami peptides, Chinese southern and northern anchovy sauce were selected for the study. Seventeen peptides were obtained by separation and purification, and their taste activity was predicted. Through the taste characterization and descriptive analysis, it was found that the synthesized peptides were umami and umami-enhancing peptides. Seventeen umami peptides were simulated and embedded into the umami receptor T1R1/T1R3 by inserting into the Venus flytrap domain (VFTD) of the T1R3 subunit; the interaction forces were mainly hydrogen bonding, electrostatic interaction, van der Waals force and hydrophobic interaction. According to the docking interaction energies, long-chain peptides may be easier to bind to the receptor than short-chain peptides. Asp196, Glu128 and Glu197 were the main binding sites for docking, and could affect umami synergism. For the first time, novel umami peptides in Chinese anchovy sauce have been reported. This study is helpful for discovering umami marine resource peptides, and can provide a basis for further understanding the flavor system of anchovy sauce. © 2020 Society of Chemical Industry.

Fluorescent pyrene moiety in fluorinated C6F5-corroles increases the interaction with HSA and CT-DNA

Two fluorinated meso-C6F5-corroles (5,15-bis(pentafluorophenyl)-10-(phenyl)corrole and 5,15-bis(pentafluorophenyl)-10-(1-pyrenyl)corrole) were biologically evaluated in terms of binding affinity to human serum albumin (HSA) and calf-thymus DNA (CT-DNA) via multiple spectroscopic techniques under physiological conditions combined with molecular docking calculations. The HSA:corrole interaction is spontaneous and moderate via static binding, disturbing both secondary and tertiary albumin structures at high fluorinated corrole concentrations. The competitive binding studies indicated positive cooperativity or allosteric activation, while molecular docking calculations suggested that both fluorinated corroles bind preferentially inside subdomains IIA and IB (sites I and III, respectively). The experimental CT-DNA binding assays indicated that fluorinated corroles interact spontaneously by non-classical modes in the minor groove of the CT-DNA strands via static fluorescence quenching mechanism. Molecular docking results also showed the minor groove as the main binding site for CT-DNA. Overall, the pyrene moiety increased the interaction with HSA and CT-DNA, which is probably due to the planarity and volume that favors the pyrene unit to be buried inside the biomacromolecule pockets.

Computational Studies towards the Identification of Novel Rhodopsin-Binding Compounds as Chemical Chaperones for Misfolded Opsins.

Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber’s congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber’s congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases.

Regulation of Properties of Lipid Membranes by Interaction with 2-Hydroxypropyl β-Cyclodextrin: Molecular Details

The interaction between 2-hydroxypropyl β-cyclodextrin (HPCD) and a liposomal bilayer has been studied. The main binding sites of HPCD on the surface of neutral liposomes based on dipalmitoylphosphatidylcholine (DPPC) are the phosphate groups of lipids. The complex formation with HPСD leads to the stabilization of the gel state of monocomponent liposomes. The inclusion of the anionic component, cardiolipin (CL, 20%), in the bilayer changes the nature of the liposome-HPCD interaction. The observed lipid disorder disturbs membrane integrity, which is revealed in the release of the included dye (phenolphthalein). The effect of HPCD on the process and parameters of the phase transition of anionic liposomes has been studied using thermograms, which show the change in the position of the absorption bands of lipid acyl chains in the FTIR spectrum of liposomes. A stratification of DPPC/CL (80/20%) bilayer into two microphases with different cardiolipin content has been detected. HPCD causes the more pronounced stratification in the bilayer, i.e., membrane destabilization near the melting point of CL-rich microphase and, vice versa, a decrease in the lipid mobility in regions with a low CL content. We have studied the effect of HPCD on the interaction of an antibacterial drug, levofloxacin (LV), with the lipid bilayer. It has been found that the complexation of the drug molecules with HPCD leads to an increase in the efficiency of drug adsorption on the surface of the bilayer. This may facilitate the transport of the drug through the bilayer due to the formation of defects in the membrane. LV in the complex with HPCD shows a high antibacterial efficiency in vitro against E. coli, which is not lower as compared with free LV.

Regions of conformational flexibility in the proprotein convertase PCSK9 and design of antagonists for LDL cholesterol lowering.

The proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol levels by binding to the liver LDL receptor (LDLR) and promoting its degradation. Therefore, PCSK9 has become a compelling new therapeutic target for lipid lowering and the prevention of cardiovascular disease. PCSK9 contains two regions of conformational flexibility, the N-terminal regions of the prodomain and of the catalytic domain. The recognition that the latter region, the so-called P' helix, is able to transition from an α-helical to a disordered state gave rise to new strategies to develop small molecule inhibitors of PCSK9 for lipid lowering. In the ordered state the P' helix is buried in a groove of the PCSK9 catalytic domain located next to the main LDLR binding site. The transition to a disordered state leaves the groove site vacated and accessible for compounds to antagonize LDLR binding. By use of a groove-directed phage display strategy we were able to identify several groove-binding peptides. Based on structural information of PCSK9-peptide complexes, a minimized groove-binding peptide was generated and utilized as an anchor to extend towards the adjacent main LDLR binding site, either by use of a phage-displayed peptide extension library, or by appending organic moieties to yield organo-peptides. Both strategies led to antagonists with pharmacologic activities in cell-based assays. The intricate bipartite mechanism of the potent organo-peptide inhibitors was revealed by structural studies, showing that the core peptide occupies the N-terminal groove, while the organic moiety interacts with the LDLR binding site to create antagonism. These findings validate the PCSK9 groove as an attractive target site and should inspire the development of a new class of small molecule antagonists of PCSK9.

Characterization of sugarcane leaf-biomass and investigation of its efficiency in removing Nickel(II), Chromium(III) and Cobalt(II) ions from polluted water

Sugarcane leaves biomass (SCL) has been utilized for the removal of Ni2+, Cr3+ and Co2+ ions from polluted water. Qualitative analysis was performed by Fourier Transformed Infra-Red spectroscopy, Raman spectroscopy, scanning electron microscopy coupled with energy dispersive spectroscopy and X-ray diffractometry. Adsorption of the metal-ions was carried out by contacting 50 mL of known concentration of Ni2+, Cr3+ and Co2+ with known amount of SCL at 27 °C, and under varied experimental conditions such as pH, ionic strength, solid-to-liquid ratio, contact time and initial metal ions concentration in batch adsorption experiment. The kinetics, isothermal, as well as the nature of the adsorption were predicted by models such as pseudo-first-order, pseudo-second order, Elovich, Weber-Morris, Freundlich, Langmuir, Temkin and Dubinin-Raduschevich. The thermodynamics of the processes was also predicted. Characterization analyses portrayed the surface of SCL as being porous, oval in shape and composed of hydroxyl and carbonyl groups as the main binding sites. Raman analysis revealed the interaction of the metal ions with the lignin, cellulose as well as hemicellulose components of the adsorbent. Adsorption of Ni2+ and Co2+ ions was favored by increasing pH, while that of Cr3+ ions was mostly favored at pH 4. Metal uptake increased with increasing contact time and concentration up to equilibrium stage. The sorption processes followed the pseudo-second-order kinetics and was monolayer in nature with qmax of 51.3, 62.5, and 66.7 mg/g for the uptake of Ni2+, Cr3+ and Co2+ ions, respectively. The models predicted physisorption as the main process involved. Thermodynamic study showed that the processes were feasible and spontaneous, endothermic, and occurred with increase in randomness at the adsorbate-adsorbent interface, demonstrating a good metal uptake nature of the adsorbent.

DNA/BSA binding of a new oxovanadium (IV) complex of glycylglycine derivative Schiff base ligand

In this study, a new oxovanadium (IV) Schiff base complex was synthesized by the reaction of {[5-(triethylammoniummethyl)salicylaldehyde]chloride}triethylammonium and vanadyl sulfate. The complex was characterized by elemental analysis, FT-IR and UV–Vis spectroscopy. The interaction between the complex and bovine serum albumin (BSA) was studied by absorption and emission titration methods. The quality of the spectral changes and the large calculated value of both the binding constant (Kb = 7.53 × 104 M−1) and the Stern-Volmer quenching constant (KSV = 7.65 × 104 M−1) proved the strong affinity of the complex to BSA. The static quenching mechanism was confirmed by comparing the calculated Kb values at four different temperatures. The negative changes in enthalpy and Gibbs free energy, proved that the BSA-complex binding is exothermic and spontaneous. The fluorescence data also indicated 1:1 binding stoichiometry of the complex on one binding site of BSA. Competitive experiments with site markers (phenylbutazone and ibuprofen) and synchronous measurements identified site I of BSA as the main binding site. Molecular docking results confirmed the binding pocket of site I (subdomain IIA) as the binding site. In the second part of the work, the binding of the complex to fish sperm DNA (FS-DNA) was investigated by UV–Vis absorption spectroscopy, fluorescence spectroscopy, viscosity measurement and cyclic voltammetry. The Stern-Volmer quenching and bimolecular quenching rate constants (KSV = 6.13 × 102 M−1, kq = 6.13 × 1010 M−1S−1) were calculated. Absorption and emission studies could present types of interactions for example groove binding and electrostatic. A minor changes in viscosity showed a groove binding mode. In cyclic voltammogram study, decrease in current and also shift of peak potentials positively after adding DNA supported a partial intercalation mode. Theoretical docking study proposed the minor groove as the main part for DNA-complex binding.

Biological assays of BF2-naphthyridine compounds: Tyrosinase and acetylcholinesterase activity, CT-DNA and HSA binding property evaluations

The present work reports the biological assays between synthetic BF2-naphtyridine complexes and four proteins: human serum albumin (HSA), calf-thymus DNA (CT-DNA), tyrosinase and acetylcholinesterase enzymes via spectroscopic analysis at physiological conditions, combined with molecular docking simulations. The BF2-complexes presented spontaneous and moderate binding ability to HSA through the ground-state association (static fluorescence quenching mechanism). The main binding site is Sudlow's site I (subdomain IIA) and the binding does not perturb significantly both secondary and surface structure of HSA. Despite BF2-complexes showed good binding ability with HSA, these compounds presented weak intercalative ability with CT-DNA (the most conventional and simple model to preliminary studies), except in the case of 1 h, which suggested that the presence of electronic donor groups in both aromatic ring moieties of BF2-complex structure can increase the intercalative ability for DNA strands. Competitive binding displacement assays in the presence of methyl green and molecular docking calculations indicated that the studied compounds interact preferentially in the major groove of DNA. In addition, the assayed compounds presented the ability to activate or inhibit both tyrosinase (the decontrolled activity can induce melanoma carcinoma) or AChE (involved in reactions related to the function of neurotransmitters) enzymes.

Acute Kidney Injury in COVID-19: Emerging Evidence of a Distinct Pathophysiology.

Acute Kidney Injury in COVID-19: Emerging Evidence of a Distinct Pathophysiology.

Structure modeling of γ-aminobutyric acid transporters – Molecular basics of ligand selectivity

γ-Aminobutyric acid transporters are responsible for regulating the GABA level in the synaptic cleft. In this way, they affect GABA-ergic transmission which is important for the proper functioning of the central nervous system. The exact structure of GABA transporters is still unknown, which hinders the design of new, potent and selective inhibitors. For these reasons, we decided to create models of all types of human gamma-aminobutyric acid transporters. They were built based on crystal structures of related proteins from the SLC6 family using homology modeling methods. The reliability of the received models has been confirmed by a number of tools assessing the quality of protein models. To determine the ligand binding mode and indicate the amino acids responsible for selectivity, docking studies and molecular dynamics simulations were performed. The amino acids lining the bottom of the main binding site have a major impact on the selective ligand binding. In addition, an important element is the non-helical fragment of the transmembrane domain 10, and several amino acids within the vestibule of the transporters, which affect its volume. To check whether obtained models are suitable to distinguish active compounds from inactive ones, enrichment plots were prepared. Results suggest that our models may be useful in the search for new inhibitors of GABA transporters of the desired selectivity.