Inhibition of phosphodiesterase 4 (PDE4) is a promising therapeutic approach for the treatment of inflammatory pulmonary disorders, i.e. asthma and chronic obstructive pulmonary disease. However, the treatment with non-selective PDE4 inhibitors is associated with side effects such as nausea and vomiting. Among the subtypes of PDE4 inhibited by these inhibitors, PDE4B is expressed in immune, inflammatory and airway smooth muscle cells, whereas, PDE4D is expressed in the area postrema and nucleus of the solitary tract. Thus, PDE4D inhibition is responsible for the emetic response. In this regard, a selective PDE4B inhibitor is expected to be a potential drug candidate for the treatment of inflammatory pulmonary disorders. Therefore, a shared feature pharmacophore model was developed and used as a query for the virtual screening of Maybridge and SPECS databases. A number of filters were applied to ensure only compounds with drug-like properties were selected. Accordingly, nine compounds have been identified as final hits, where HTS04529 showed the highest affinity and selectivity for PDE4B over PDE4D in molecular docking. The docked complexes of HTS04529 with PDE4B and PDE4D were subjected to molecular dynamics simulations for 100ns to assess their binding stability. The results showed that HTS04529 was bound tightly to PDE4B and formed a more stable complex with it than with PDE4D.
The influence of methanol as a solvent on the properties of sodium alginate/sulfonated graphene oxide (SA/SGO) membranes was explored in water-methanol mixed conditions with various methanol concentrations and temperatures through molecular dynamics simulations. The methanol uptake of the membrane showed an isolation phase determined from the simulation results. The distance between the sulfonic acid groups increased in higher methanol concentrations, as observed from S-S RDFs. Furthermore, the distance between the SA-chain RDFs and the solvent molecules was analysed to determine a) the affinity of water towards the sulfonic acid groups and b) the affinity of the aromatic backbone of the SA towards methanol molecules. A decrease in water molecule diffusion led to an increase in methanol diffusion and uptake. SA/SGO membranes exhibited a smaller diffusion coefficient than that for the Nafion membranes, as calculated from simulation results and compared to the experimental work. Additionally, the diffusion ability increased at higher temperatures for all permeants. The interaction information obtained is useful for DMFC applications.
Dengue infections are currently estimated to be 390 million cases annually. Yet, there is no vaccine or specific therapy available. Envelope glycoprotein E (E protein) of DENV mediates viral attachment and entry into the host cells. Several flavonoids have been shown to inhibit HIV-1 and hepatitis C virus entry during the virus-host membrane fusion. In this work, molecular docking method was employed to predict the binding of nine flavonoids (baicalin, baicalein, EGCG, fisetin, glabranine, hyperoside, ladanein, quercetin and flavone) to the soluble ectodomain of DENV type 2 (DENV2) E protein. Interestingly, eight flavonoids were found to dock into the same binding pocket located between the domain I and domain II of different subunits of E protein. Consistent docking results were observed not only for the E protein structures of the DENV2-Thai and DENV2-Malaysia (a homology model) but also for the E protein structures of tick-borne encephalitis virus and Japanese encephalitis virus. In addition, molecular dynamics simulations were performed to further evaluate the interaction profile of the docked E protein-flavonoid complexes. Ile4, Gly5, Asp98, Gly100 and Val151 residues of the DENV2-My E protein that aligned to the same residues in the DENV2-Thai E protein form consistent hydrogen bond interactions with baicalein, quercetin and EGCG during the simulations. This study demonstrates flavonoids potentially form interactions with the E protein of DENV2.
We report a detailed structural analysis of the psychrophilic exo-β-1,3-glucanase (GaExg55) from Glaciozyma antarctica PI12. This study elucidates the structural basis of exo-1,3-β-1,3-glucanase from this psychrophilic yeast. The structural prediction of GaExg55 remains a challenge because of its low sequence identity (37 %). A 3D model was constructed for GaExg55. Threading approach was employed to determine a suitable template and generate optimal target-template alignment for establishing the model using MODELLER9v15. The primary sequence analysis of GaExg55 with other mesophilic exo-1,3-β-glucanases indicated that an increased flexibility conferred to the enzyme by a set of amino acids substitutions in the surface and loop regions of GaExg55, thereby facilitating its structure to cold adaptation. A comparison of GaExg55 with other mesophilic exo-β-1,3-glucanases proposed that the catalytic activity and structural flexibility at cold environment were attained through a reduced amount of hydrogen bonds and salt bridges, as well as an increased exposure of the hydrophobic side chains to the solvent. A molecular dynamics simulation was also performed using GROMACS software to evaluate the stability of the GaExg55 structure at varying low temperatures. The simulation result confirmed the above findings for cold adaptation of the psychrophilic GaExg55. Furthermore, the structural analysis of GaExg55 with large catalytic cleft and wide active site pocket confirmed the high activity of GaExg55 to hydrolyze polysaccharide substrates.
The utilization of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages. However, an adaptation of enzyme towards organic solvent is unpredictable and not fully understood because of limited information on the organic solvent tolerant enzymes. To understand how the enzyme can adapt to the organic solvent environment, structural and computational approaches were employed. A recombinant elastase from Pseudomonas aeruginosa strain K was an organic solvent tolerant zinc metalloprotease was successfully crystallized and diffracted up to 1.39 Å. Crystal structure of elastase from strain K showed the typical, canonical alpha-beta hydrolase fold consisting of 10-helices (118 residues), 10- β-strands (38 residues) and 142 residues were formed other secondary structure such as loop and coil to whole structure. The elastase from Pseusomonas aeruginosa strain K possess His-140, His-144 and Glu-164 served as a ligand for zinc ion. The conserved catalytic triad was composed of Glu-141, Tyr-155 and His-223. Three-dimensional structure features such as calcium-binding and presence of disulphide-bridge contribute to the stabilizing the elastase structure. Molecular dynamic (MD) simulation of elastase revealed that, amino acid residues located at the surface area and disulphide bridge in Cys-30 to Cys-58 were responsible for enzyme stability in organic solvents.
In this study, caffeine (CA) was encapsulated into food-grade starch matrices, including swelled starch (SS), porous starch (PS), and V-type starch (VS). The bitterness of the microcapsules and suppression mechanisms were investigated using an electronic tongue, molecular dynamics (MD) simulation and the in vitro release kinetics of CA. All the CA-loaded microcapsules showed a lower bitterness intensity than the control. The MD results proved that the weak interactions between starch and CA resulted in a moderate CA release rate for SS-CA microcapsules. The PS-CA microcapsule presented the longest CA release, up to 40 min, whereas the VS-CA microcapsule completely released CA in 9 min. The CA release rate was found to be related to the microcapsule structure and rehydration properties. A low CA bitterness intensity could be attributed to a delay in the CA release rate and resistance to erosion of the microcapsules. The results of this work are valuable for improving starch-based microcapsules (oral-targeted drug-delivery systems) by suppressing the bitterness of alkaloid compounds.
Genome data mining of the Antarctic yeast, Glaciozyma antarctica PI12 revealed an expansin-like protein encoding sequence (GaEXLX1). The GaEXLX1 protein is 24.8 kDa with a high alkaline pI of 9.81. Homology modeling of GaEXLX1 showed complete D1 and D2 domains of a conventional expansin. The protein exhibited 36% sequence similarity to Clavibacter michiganensis EXLX1 (PDB: 4JCW). Subsequently, a recombinant GaEXLX1 protein was produced using Escherichia coli expression system. Incubation with Avicel, filter paper and cotton fiber showed that the protein can disrupt the surface of crystalline and pure cellulose, suggesting a cell wall modification activity usually exhibited by expansin-like proteins. Binding assays displayed that GaEXLX1 can bind to polymeric substrates, including those postulated to be present in the sea ice ecosystem such as crab chitin and moss lichenan. GaEXLX1 may assist in the recognition and loosening of these substrates in the sea ice prior to hydrolysis by other extracellular enzymes. Similar loosening mechanism to classical expansin-like protein has been postulated for this psychrophilic protein based on several conserved residues of GaEXLX1 involved in binding interaction identified by docking analyses.
Jamestown Canyon virus (JCV) is a deadly viral infection transmitted by various mosquito species. This mosquito-borne virus belongs to Bunyaviridae family, posing a high public health threat in the in tropical regions of the United States causing encephalitis in humans. Common symptoms of JCV include fever, headache, stiff neck, photophobia, nausea, vomiting, and seizures. Despite the availability of resources, there is currently no vaccine or drug available to combat JCV. The purpose of this study was to develop an epitope-based vaccine using immunoinformatics approaches. The vaccine aimed to be secure, efficient, bio-compatible, and capable of stimulating both innate and adaptive immune responses. In this study, the protein sequence of JCV was obtained from the NCBI database. Various bioinformatics methods, including toxicity evaluation, antigenicity testing, conservancy analysis, and allergenicity assessment were utilized to identify the most promising epitopes. Suitable linkers and adjuvant sequences were used in the design of vaccine construct. 50s ribosomal protein sequence was used as an adjuvant at the N-terminus of the construct. A total of 5 CTL, 5 HTL, and 5 linear B cell epitopes were selected based on non-allergenicity, immunological potential, and antigenicity scores to design a highly immunogenic multi-peptide vaccine construct. Strong interactions between the proposed vaccine and human immune receptors, i.e., TLR-2 and TLR-4, were revealed in a docking study using ClusPro software, suggesting their possible relevance in the immunological response to the vaccine. Immunological and physicochemical properties assessment ensured that the proposed vaccine demonstrated high immunogenicity, solubility and thermostability. Molecular dynamics simulations confirmed the strong binding affinities, as well as dynamic and structural stability of the proposed vaccine. Immune simulation suggest that the vaccine has the potential to effectively stimulate cellular and humoral immune responses to combat JCV infection. Experimental and clinical assays are required to validate the results of this study.
This work employed the model protein β-lactoglobulin (BLG) to investigate the contribution of microstructural changes to regulating the interaction patterns between protein and flavor compounds through employing computer simulation and multi-spectroscopic techniques. The formation of molten globule (MG) state-like protein during the conformational evolution of BLG, in response to ultrasonic (UC) and heat (HT) treatments, was revealed through multi-spectroscopic characterization. Differential MG structures were distinguished by variations in surface hydrophobicity and the microenvironment of tryptophan residues. Fluorescence quenching measurements indicated that the formation of MG enhanced the binding affinity of heptanal to protein. LC-MS/MS and NMR revealed the covalent bonding between heptanal and BLG formed by Michael addition and Schiff-base reactions, and MG-like BLG exhibited fewer chemical shift residues. Molecular docking and molecular dynamics simulation confirmed the synergistic involvement of hydrophobic interactions and hydrogen bonds in shaping BLG-heptanal complexes thus promoting the stability of BLG structures. These findings indicated that the production of BLG-heptanal complexes was driven synergistically by non-covalent and covalent bonds, and their interaction processes were influenced by processes-induced formation of MG potentially tuning the release and retention behaviors of flavor compounds.
Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.
Currently, there is no three-dimensional structure of D-specific dehalogenase (DehD) in the protein database. We modeled DehD using ab initio technique, performed molecular dynamics (MD) simulation and docking of D-2-chloropropionate (D-2CP), D-2-bromopropionate (D-2BP), monochloroacetate (MCA), monobromoacetate (MBA), 2,2-dichloropropionate (2,2-DCP), d,l-2,3-dichloropropionate (d,l-2,3-DCP), and 3-chloropropionate (3-CP) into the DehD active site. The sequences of DehD and D-2-haloacid dehalogenase (HadD) from Pseudomonas putida AJ1 have 15% sequence similarity. The model had 80% of the amino acid residues in the most favored region when compared to the crystal structure of DehI from Pseudomonas putida PP3. Docking analysis revealed that Arg107, Arg134 and Tyr135 interacted with D-2CP, and Glu20 activated the water molecule for hydrolytic dehalogenation. Single residue substitutions at 25-30 °C showed that polar residues of DehD were stable when substituted with nonpolar residues and showed a decrease in activity within the same temperature range. The molecular dynamics simulation of DehD and its variants showed that in R134A variant, Arg107 interacted with D-2CP, while in Y135A, Gln221 and Arg231 interacted with D-2CP. It is our emphatic belief that the new model will be useful for the rational design of DehDs with enhanced potentials.
Fevicordin-A (FevA) isolated from Phaleria macrocarpa (Scheff) Boerl. seeds was evaluated for its potential anticancer activity by in vitro and in silico approaches. Cytotoxicity studies indicated that FevA was selective against cell lines of human breast adenocarcinoma (MCF-7) with an IC50 value of 6.4 µM. At 11.2 µM, FevA resulted in 76.8% cell death of T-47D human breast cancer cell lines. Critical pharmacophore features amongst human Estrogen Receptor-α (hERα) antagonists were conserved in FevA with regard to a hypothesis that they could make notable contributions to its pharmacological activity. The binding stability as well as the dynamic behavior of FevA towards the hERα receptor in agonist and antagonist binding sites were probed using molecular dynamics (MD) simulation approach. Analysis of MD simulation suggested that the tail of FevA was accountable for the repulsion of the C-terminal of Helix-11 (H11) in both agonist and antagonist receptor forms. The flexibility of loop-534 indicated the ability to disrupt the hydrogen bond zipper network between H3 and H11 in hERα. In addition, MM/GBSA calculation from the molecular dynamic simulations also revealed a stronger binding affinity of FevA in antagonistic action as compared to that of agonistic action. Collectively, both the experimental and computational results indicated that FevA has potential as a candidate for an anticancer agent, which is worth promoting for further preclinical evaluation.
Lipase plays an important role in industrial and biotechnological applications. Lipases have been subject to modification at the N and C terminals, allowing better understanding of lipase stability and the discovery of novel properties. A thermotolerant lipase has been isolated from Antarctic Pseudomonas sp. The purified Antarctic AMS3 lipase (native) was found to be stable across a broad range of temperatures and pH levels. The lipase has a partial Glutathione-S-transferase type C (GST-C) domain at the N-terminal not found in other lipases. To understand the influence of N-terminal GST-C domain on the biochemical and structural features of the native lipase, the deletion of the GST-C domain was carried out. The truncated protein was successfully expressed in E. coli BL21(DE3). The molecular weight of truncated AMS3 lipase was approximately ~45 kDa. The number of truncated AMS3 lipase purification folds was higher than native lipase. Various mono and divalent metal ions increased the activity of the AMS3 lipase. The truncated AMS3 lipase demonstrated a similarly broad temperature range, with the pH profile exhibiting higher activity under alkaline conditions. The purified lipase showed a substrate preference for a long carbon chain substrate. In addition, the enzyme activity in organic solvents was enhanced, especially for toluene, Dimethylsulfoxide (DMSO), chloroform and xylene. Molecular simulation revealed that the truncated lipase had increased structural compactness and rigidity as compared to native lipase. Removal of the N terminal GST-C generally improved the lipase biochemical characteristics. This enzyme may be utilized for industrial purposes.
Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in "Brugia Rapid". However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.
Palm oil-based esters (POEs) are unsaturated and non-ionic esters with a great potential to act as chemical penetration enhancers and drug carriers for transdermal drug nano-delivery. A ratio of palmitate ester and nonionic Tween80 with and without diclofenac acid was chosen from an experimentally determined phase diagram. Molecular dynamics simulations were performed for selected compositions over a period of 15 ns. Both micelles showed a prolate-like shape, while adding the drug produced a more compact micellar structure. Our results proposed that the drug could behave as a co-surfactant in our simulated model.
Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use.
In this work we assessed the suitability of two different lipid membranes for the simulation of a TolC protein from Salmonella enterica serovar Typhi. The TolC protein family is found in many pathogenic Gram-negative bacteria including Vibrio cholera and Pseudomonas aeruginosa and acts as an outer membrane channel for expulsion of drug and toxin from the cell. In S. typhi, the causative agent for typhoid fever, the TolC outer membrane protein is an antigen for the pathogen. The lipid environment is an important modulator of membrane protein structure and function. We evaluated the conformation of the TolC protein in the presence of DMPE and POPE bilayers using molecular dynamics simulation. The S. typhi TolC protein exhibited similar conformational dynamics to TolC and its homologues. Conformational flexibility of the protein is seen in the C-terminal, extracellular loops, and α-helical region. Despite differences in the two lipids, significant similarities in the motion of the protein in POPE and DMPE were observed, including the rotational motion of the C-terminal residues and the partially open extracellular loops. However, analysis of the trajectories demonstrated effects of hydrophobic matching of the TolC protein in the membrane, particularly in the lengthening of the lipids and subtle movements of the protein's β-barrel towards the lower leaflet in DMPE. The study exhibited the use of molecular dynamics simulation in revealing the differential effect of membrane proteins and lipids on each other. In this study, POPE is potentially a more suitable model for future simulation of the S. typhi TolC protein.
Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB-HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92-6.89 × 10(3 )M(-1) at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB-HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J mol(-1) K(-1)) and negative ΔH (-6.57 kJ mol(-1)) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB-HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow's site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca(2+), Zn(2+), Cu(2+), Ba(2+), Mg(2+), and Mn(2+) in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.
Vascular endothelial growth factor (VEGF) and its co-receptor neuropilin-1 (NRP-1) are important targets of many pro-angiogenic factors. In this study, nine peptides were synthesized and evaluated for their molecular interaction with NRP-1 and compared to our previous peptide ATWLPPR. Docking study showed that the investigated peptides shared the same binding region as shown by tuftsin known to bind selectively to NRP-1. Four pentapeptides (DKPPR, DKPRR, TKPPR and TKPRR) and a hexapeptide CDKPRR demonstrated good inhibitory activity against NRP-1. In contrast, peptides having arginine residue at sites other than the C-terminus exhibited low activity towards NRP-1 and this is confirmed by their inability to displace the VEGF165 binding to NRP-1. Docking study also revealed that replacement of carboxyl to amide group at the C-terminal arginine of the peptide did not affect significantly the binding interaction to NRP-1. However, the molecular affinity study showed that these peptides have marked reduction in the activity against NRP-1. Pentapeptides having C-terminal arginine showed strong interaction and good inhibitory activity with NRP thus may be a good template for anti-angiogenic targeting agent.
Paenibacillus polymyxa β-glucosidase B (BglB), belongs to a GH family 1, is a monomeric enzyme that acts as an exo-β-glucosidase hydrolysing cellobiose and cellodextrins of higher degree of polymerization using retaining mechanism. A molecular dynamics (MD) simulation was performed at 300 K under periodic boundary condition for 5 ns using the complexes structure obtained from previous docking study, namely BglB-Beta-d-glucose and BglB-Cellobiose. From the root-mean-square deviation analysis, both enzyme complexes were reported to deviate from the initial structure in the early part of the simulation but it was stable afterwards. The root-mean-square fluctuation analysis revealed that the most flexible regions comprised of the residues from 26 to 29, 43 to 53, 272 to 276, 306 to 325 and 364 to 367. The radius of gyration analysis had shown the structure of BglB without substrate became more compact towards the end of the simulation compare to other two complexes. The residues His122 and Trp410 were observed to form stable hydrogen bond with occupancy higher than 10%. In conclusion, the behaviour of BglB enzyme towards the substrate binding was successfully explored via MD simulation approaches.