Structure of glutathione S-transferase of the filarial parasite Wuchereria bancrofti: a target for drug development against adult worm

A three dimensional structural model of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. The three dimensional X-ray crystal structure of porcine -class GST with PDB ID: 2gsr-A chain protein with 42% sequential and functional homology was used as the template. The model of wbGST built by MODELLER6v2 was analyzed by the PROCHECK programs. Ramachandran plot analysis showed that 93.5% of the residues are in the core region followed by 5.4 and 1.1% residues in the allowed and generously allowed regions, respectively. None of the non-glycine residues is in disallowed regions. The PROSA II z-score and the energy graph for the final model further confirmed the quality of the modeled structure. The computationally modeled three-dimensional (3D) structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668). 1SFM was used for docking with GST inhibitors by Hex4.2 macromolecular docking using spherical polar Fourier correlations.Figure: A three-dimensional (3D) structure of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. This modeled 3D structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668).     

Three-dimensional model of a substrate-bound SARS chymotrypsin-like cysteine proteinase predicted by multiple molecular dynamics simulations: catalytic efficiency regulated by substrate binding

Severe acute respiratory syndrome (SARS) is a contagious and deadly disease caused by a new coronavirus. The protein sequence of the chymotrypsin-like cysteine proteinase (CCP) responsible for SARS viral replication has been identified as a target for developing anti-SARS drugs. Here, I report the ATVRLQ(p1)A(p1')-bound CCP 3D model predicted by 420 different molecular dynamics simulations (2.0 ns for each simulation with a 1.0-fs time step). This theoretical model was released at the Protein Data Bank (PDB; code: 1P76) before the release of the first X-ray structure of CCP (PDB code: 1Q2W). In contrast to the catalytic dyad observed in X-ray structures of CCP and other coronavirus cysteine proteinases, a catalytic triad comprising Asp187, His41, and Cys145 is found in the theoretical model of the substrate-bound CCP. The simulations of the CCP complex suggest that substrate binding leads to the displacement of a water molecule entrapped by Asp187 and His41, thus converting the dyad to a more efficient catalytic triad. The CCP complex structure has an expanded active-site pocket that is useful for anti-SARS drug design. In addition, this work demonstrates that multiple molecular dynamics simulations are effective in correcting errors that result from low-sequence-identity homology modeling.     

Molecular cloning, sequence analysis and structure modeling of OmpR, the response regulator of Aeromonas hydrophila

The ability of bacteria to survive and proliferate in changing environmental conditions, and during host cell invasion is the key to their pathogenicity. In order to achieve this, the bacteria use a signal transduction system, the two component regulatory system, which consists of a sensor kinase and a response regulator. The EnvZ/OmpR system regulates the porin genes ompF/ompC in response to changes in osmolarity. In the present study, the ompR gene of Aeromonas hydrophila (isolate Ah17) was cloned, sequenced and characterized. Further an attempt was made to analyze the structural characteristics of the OmpR protein from Aeromonas hydrophila. The three dimensional structure of the protein was predicted by homology modeling and the modeled structure was compared to other members of two component response regulators. This study would be helpful for structure based drug design approaches to generate drugs against this harmful pathogen to control its proliferation in both human and fish hosts.     

黄芩苷对嗜水气单胞菌生物膜形成的影响及体内外的抑菌作用

[目的]探讨中药单体黄芩苷对嗜水气单胞菌在体内外生长及生物膜形成的影响.[方法]体外实验中,利用牛津杯法检测抑菌圈直径,结晶紫法检测生物膜的形成,通过泳动实验检测黄芩苷对嗜水气单胞菌运动性的影响,紫外吸收法检测细胞膜完整性,用透射电镜技术观察黄芩苷对细菌形态的影响.体内实验利用草鱼为对象检测黄芩苷对嗜水气单胞菌增殖的影...     

Discovery of 6-benzyloxyquinolines as c-Met selective kinase inhibitors

A novel quinoline derivative that selectively inhibits c-Met kinase was identified. The molecular design is based on a result of the analysis of a PF-2341066 (1)/c-Met cocrystal structure (PDB code: 2wgj). The kinase selectivity of the derivatives is discussed from the view point of the sequence homology of the kinases, the key interactions found in X-ray cocrystal structures, and the structure–activity relationship (SAR) obtained in this work.     

Online homology modelling as a means of bridging the sequence-structure gap

For even the best-studied species, there is a large gap in their representation in the protein databank (PDB) compared to within sequence databases. Typically, less than 2% of sequences are represented in the PDB. This is partly due to the considerable experimental challenge and manual inputs required to solve three dimensional structures by methods such as X-ray diffraction and multi-dimensional nuclear magnetic resonance (NMR) spectroscopy in comparison to high-throughput sequencing. This gap is made even wider by the high level of redundancy within the PDB and under-representation of some protein categories such as membrane-associated proteins which comprise approximately 25% of proteins encoded in genomes. A traditional route to closing the sequence-structure gap is offered by homology modelling whereby the sequence of a target protein is modelled on a template represented in the PDB using in silico energy minimisation approaches. More recently, online homology servers have become available which automatically generate models from proffered sequences. However, many online servers give little indication of the structural plausibility of the generated model. In this paper, the online homology server Geno3D will be described. This server uses similar software to that used in modelling structures during structure determination and thus generates data allowing determination of the structural plausibility of models. For illustration, modelling of a chemotaxis protein (CheY) from Pseudomononas entomophila L48 (accession YP_609298) on a template (PDB id. 1mvo), the phosphorylation domain of an outer membrane protein PhoP from Bacillus subtilis, will be described.     

Three dimensional modeling of C-terminal loop of CssA subunit in CS6 of Enterotoxigenic Escherichia coli and its interaction with the 70 KDa domain of Fibronectin

Colonization factor CS6 of enterotoxigenic Escherichia coli (ETEC) helps to establish the adherence of CS6-expressing ETEC in the intestinal wall. CS6 is composed of two structural subunits, known as CssA and CssB. During CS6-expressing ETEC adherence in intestinal wall, 15 amino acid residues containing Cterminal region of CssA subunit, help to bind with N-terminal 70kDa domain of fibronectin (Fn). In this study, we have predicted a theoretical structural model for C-terminal domain of CssA by homology modelling using protein data bank (PDB) file, 1NTY-A as template (66.67% sequence identity) in Discovery Studio. The structural model of N-terminal region of Fn was also determined by homology modelling using PDB files 1FBR and 1E88 as templates. The structure of the model was also validated by Ramachandran plot. The energy minimization for Fn was performed in standard dynamic cascade using Steepest Descent algorithm followed by Adopted Basis NR algorithm in Discovery studio. The docking model between C-terminal domain and fibronectin were generated by using ClusPro algorithm. This docking study would be help for better understanding how CS6 interacts with fibronectin of intestinal extracellular matrix in the host during infection, and would be of great help towards subunit vaccine generation.     

Molecular detection and cloning of thermostable hemolysin gene from <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Aeromonas hydrophila is a major bacterial pathogen associated with hemorrhagic septicemia in aquatic and terrestrial animals including humans. There is an urgent need to develop molecular and immunological assays for rapid, specific and sensitive diagnosis. A new set of primers has been designed for detection of thermostable hemolysin (TH) gene (645 bp) from A. hydrophila, and sensitivity limit for detection of TH gene was 5 pg. The TH gene was cloned, sequenced and analyzed. The G+C content was 68.06%; and phylogeny was constructed using TH protein sequences which had significant homology with those for thermostable and other hemolysins present in several bacterial pathogens. In addition, we have predicted the four and eight T-cell epitopes for MHC class I and II alleles, respectively. These results provide new insight for TH protein containing antigenic epitopes that can be used in immunoassays and also designing of thermostable vaccines.     

Structural insights into human GPCR protein OA1: a computational perspective

Human ocular albinism type 1 protein (OA1)—a member of the G-protein coupled receptor (GPCR) superfamily—is an integral membrane glycoprotein expressed exclusively by intracellular organelles known as melanocytes, and is responsible for the proper biogenesis of melanosomes. Mutations in the Oa1 gene are responsible for the disease ocular albinism. Despite its clinical importance, there is a lack of in-depth understanding of its structure and mechanism of activation due to the absence of a crystal structure. In the present study, homology modeling was applied to predicting OA1 structure following thorough sequence analysis and secondary structure predictions. The predicted model had the signature residues and motifs expected of GPCRs, and was used for carrying out molecular docking studies with an endogenous ligand, l-DOPA and an antagonist, dopamine; the results agreed quite well with the available experimental data. Finally, three sets of explicit molecular dynamics simulations were carried out in lipid bilayer, the results of which not only confirmed the stability of the predicted model, but also helped witness some differences in structural features such as rotamer toggle switch, helical tilts and hydrogen bonding pattern that helped distinguish between the agonist- and antagonist-bound receptor forms. In place of the typical “D/ERY”-motif-mediated “ionic lock”, a hydrogen bond mediated by the “DAY” motif was observed that could be used to distinguish the agonist and antagonist bound forms of OA1. In the absence of a crystal structure, this study helped to shed some light on the structural features of OA1, and its behavior in the presence of an agonist and an antagonist, which might be helpful in the future drug discovery process for ocular albinism.     

Decolorization of polymeric dyes by a novel Penicillium isolate

A novel polymeric dye-degrading fungal strain ATCC 74414 was isolated. Taxonomic identification including morphological and cultural characterization indicated that this isolate was a strain of Penicillium. Strain ATCC 74414 aerobically decolorized both Poly R-478 and Poly S-119 in liquid media containing 0.01% of polymeric dyes. The decolorization rate was examined in three distinct liquid media: Schenk and Hildebrandt-K₂SO₄ medium (SHK), potato dextrose broth (PDB), and half Murashige-Skoog medium (HMS). Strain ATCC 74414 rapidly decolorized R-478 in SHK medium but the color was subsequently released from the mycelial mass into the medium after 2–3 days, indicating that the decolorization in SHK medium could be due to adsorption of Poly R-478 by the mycelia. In contrast, in HMS and PDB media ATCC 74414 decolorized Poly R-478 more steadily, and the dye was initially adsorbed onto the mycelia and was subsequently decolorized without being released into the medium. Strain ATCC 74414 also decolorized Poly S-119 steadily in SHK, HMS and PDB media. It appears that the decolorization process involved initial mycelial adsorption of dye compounds, which was probably followed by biodegradation through microbial metabolism, and the decolorization may be affected by medium constituents. Although aerobic decolorization may not necessarily lead to complete mineralization of dyes, these results have suggested the potential of strain ATCC 74414 in bioremediation of dye-contaminated water and soil.     

Crystal structure of a putative isochorismatase hydrolase from Oleispira antarctica

Isochorismatase-like hydrolases (IHL) constitute a large family of enzymes divided into five structural families (by SCOP). IHLs are crucial for siderophore-mediated ferric iron acquisition by cells. Knowledge of the structural characteristics of these molecules will enhance the understanding of the molecular basis of iron transport, and perhaps resolve which of the mechanisms previously proposed in the literature is the correct one. We determined the crystal structure of the apo-form of a putative isochorismatase hydrolase OaIHL (PDB code: 3LQY) from the antarctic γ-proteobacterium Oleispira antarctica, and did comparative sequential and structural analysis of its closest homologs. The characteristic features of all analyzed structures were identified and discussed. We also docked isochorismate to the determined crystal structure by in silico methods, to highlight the interactions of the active center with the substrate. The putative isochorismate hydrolase OaIHL from O. antarctica possesses the typical catalytic triad for IHL proteins. Its active center resembles those IHLs with a D–K–C catalytic triad, rather than those variants with a D–K–X triad. OaIHL shares some structural and sequential features with other members of the IHL superfamily. In silico docking results showed that despite small differences in active site composition, isochorismate binds to in the structure of OaIHL in a similar mode to its binding in phenazine biosynthesis protein PhzD (PDB code 1NF8).     

Ligand-supported homology modeling of the human angiotensin II type 1 (AT(1)) receptor: insights into the molecular determinants of telmisartan binding

Angiotensin II type 1 (AT(1)) receptor belongs to the super-family of G-protein-coupled receptors, and antagonists of the AT(1) receptor are effectively used in the treatment of hypertension. To understand the molecular interactions of these antagonists, such as losartan and telmisartan, with the AT(1) receptor, a homology model of the human AT(1) (hAT(1)) receptor with all connecting loops was constructed from the 2.6 A resolution crystal structure (PDB i.d., 1L9H) of bovine rhodopsin. The initial model generated by MODELLER was subjected to a stepwise ligand-supported model refinement. This protocol involved initial docking of non-peptide AT(1) antagonists in the putative binding site, followed by several rounds of iterative energy minimizations and molecular dynamics simulations. The final model was validated based on its correlation with several structure-activity relationships and site-directed mutagenesis data. The final model was also found to be in agreement with a previously reported AT(1) antagonist pharmacophore model. Docking studies were performed for a series of non-peptide AT(1) receptor antagonists in the active site of the final hAT(1) receptor model. The docking was able to identify key molecular interactions for all the AT(1) antagonists studied. Reasonable correlation was observed between the interaction energy values and the corresponding binding affinities of these ligands, providing further validation for the model. In addition, an extensive unrestrained molecular dynamics simulation showed that the docking-derived bound pose of telmisartan is energetically stable. Knowledge gained from the present studies can be used in structure-based drug design for developing novel ligands for the AT(1) receptor.     

An interrupted beta-propeller and protein disorder: structural bioinformatics insights into the N-terminus of alsin

Defects in the human ALS2 gene, which encodes the 1,657-amino-acid residue protein alsin, are linked to several related motor neuron diseases. We created a structural model for the N-terminal 690-residue region of alsin through comparative modelling based on regulator of chromosome condensation 1 (RCC1). We propose that this alsin region contains seven RCC1-like repeats in a seven-bladed beta-propeller structure. The propeller is formed by a double clasp arrangement containing two segments (residues 1–218 and residues 525–690). The 306-residue insert region, predicted to lie within blade 5 and to be largely disordered, is poorly conserved across species. Surface patches of evolutionary conservation probably indicate locations of binding sites. Both disease-causing missense mutations—Cys157Tyr and Gly540Glu—are buried in the propeller and likely to be structurally disruptive. This study aids design of experimental studies by highlighting the importance of construct length, will enhance interpretation of protein–protein interactions, and enable rational site-directed mutagenesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Crystallographic structure versus homology model: a case study of molecular dynamics simulation of human and zebrafish histone deacetylase 10

Abstract

Histone deacetylase (HDAC) 10 has been implicated in the pathology of various cancers and neurodegenerative disorders, making the discovery of novel inhibitors of the isoform an important endeavor. However, the unavailability of crystallographic structure of human HDAC10 (hHDAC10) hinders structure-based drug design effort. Previously, we reported the homology modeled structure of human HDAC10 built using the crystallographic structure of Danio rerio (zebrafish) HDAC10 (zHDAC10) (Protein Data Bank (PDB) ID; 5TD7, released on 24 May 2017) as a template. Here, in continuation with our study, both hHDAC10 and zHDAC10, and their respective complexes with trichostatin A (TSA), quisinostat, and the native ligand (in 5TD7), 7-[(3-aminopropyl)amino]-1,1,1-trifluoroheptane-2,2-diol (PDB ID; FKS) were submitted to 100?ns-long unrestrained molecular dynamics (MD) simulations. Comparative analyses of the MD trajectories revealed that zHDAC10 and its complexes displayed higher stability than hHDAC10 and its corresponding complexes over time. Nonetheless, docking of active and inactive set molecules revealed that more reliable conformations of hHDAC10 could be obtained at an extended time period. This study may shed more light on the reliability of hHDAC10 modeled structure for use in selective inhibitor design.

Communicated by Ramaswamy H. Sarma     

The solution structure of the core of mesoderm development (MESD), a chaperone for members of the LDLR-family

Mesoderm development (MESD) is a 224 amino acid mouse protein that acts as a molecular chaperone for receptors of the low-density lipoprotein receptor (LDLR) family. By recording ¹⁵N-HSQC-NMR spectra of six different MESD constructs, we could determine a highly structured core region corresponding to residues 104-177. Here we firstly present the solution structure of this highly conserved core of MESD. It shows a four-stranded anti-parallel β-sheet and two α-helices situated on one side of the sheet. Although described in the literature as structurally homologues to ferredoxins, the connectivity of secondary structure elements is different in the MESD fold. A structural comparison to entries of the PDB reveals a frequent domain with low sequence homology annotated as HMA and P-II domains in Pfam. Accession Numbers: The structure coordinates of MESD89-184 have been deposited in the Protein Data Bank under the accession code 2I9S     

Structure-based design of a novel series of azetidine inhibitors of the hepatitis C virus NS3/4A serine protease

Structural homology between thrombin inhibitors and the early tetrapeptide HCV protease inhibitor led to the bioisosteric replacement of the P2 proline by a 2,4-disubstituted azetidine within the macrocyclic β-strand mimic. Molecular modeling guided the design of the series. This approach was validated by the excellent activity and selectivity in biochemical and cell based assays of this novel series and confirmed by the co-crystal structure of the inhibitor with the NS3/4A protein (PDB code: 4TYD).     

Homology modeling and assigned functional annotation of an uncharacterized antitoxin protein from Streptomyces xinghaiensis

Streptomyces xinghaiensis is a Gram-positive, aerobic and non-motile bacterium. The bacterial genome is known. Therefore, it is of interest to study the uncharacterized proteins in the genome. An uncharacterized protein (gi|518540893|86 residues) in the genome was selected for a comprehensive computational sequence-structure-function analysis using available data and tools. Subcellular localization of the targeted protein with conserved residues and assigned secondary structures is documented. Sequence homology search against the protein data bank (PDB) and non-redundant GenBank proteins using BLASTp showed different homologous proteins with known antitoxin function. A homology model of the target protein was developed using a known template (PDB ID: 3CTO:A) with 62% sequence similarity in HHpred after assessment using programs PROCHECK and QMEAN6. The predicted active site using CASTp is analyzed for assigned anti-toxin function. This information finds specific utility in annotating the said uncharacterized protein in the bacterial genome.